JP7516613B2 - Apolipoprotein C3 (APOC3) iRNA Compositions and Methods of Use Thereof - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/080,941, filed November 17, 2014, and U.S. Provisional Patent Application No. 62/136,159, filed March 20, 2015. The entire contents of each of the foregoing applications are hereby incorporated by reference herein.

SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 17, 2015, is named 121301-02520_SL.txt, and is 212,085 bytes in size.

Apolipoprotein C3 (APOC3) is a very low density lipoprotein (VLDL) and key regulator of lipoprotein metabolism. In humans, APOC2 is encoded by the APOC3 gene, which is located in a gene cluster with the APOA1 and APOA4 genes on the long arm of chromosome 11. APOC3 is expressed as a small 99 amino acid protein in the liver and, to a lesser extent, in the intestine. Removal of the 20 amino acid signal peptide in the endoplasmic reticulum results in the formation of the 79 amino acid mature ApoC3 protein, which can exist as a non-glycosylated or glycosylated isoform.

The main role of APOC3 is as a regulator of lipolysis by noncompetitive inhibition of endothelial-bound lipoprotein lipase (LPL). LPL hydrolyzes triacylglycerols in triacylglycerol-rich lipoproteins (TRLs) to liberate fatty acids in plasma and converts large triacylglycerol-rich particles into small triacylglycerol-depleted remnant lipoproteins. APOC3 deficient individuals have low TRL levels, accompanied by highly efficient lipolysis of triacylglycerols. Furthermore, mice genetically deleted for the APOC3 gene have also been shown to have low plasma triacylglycerol levels and efficient TRL catabolism. APOC3 also inhibits hepatic lipase (HL), a lipolytic enzyme with triacylglycerol lipase and phospholipase A1 activity that is synthesized in the liver. The HL inhibitory effect of APOC3 further reduces lipolysis and uptake of TRL remnants by the liver. APOC3 has also been shown to stimulate the synthesis of very low density lipoprotein (VLDL). The underlying mechanism associated with this APOC3 effect may involve inhibition of proteasome-mediated APOB degradation, leading to increased APOB synthesis and secretion, as well as increased synthesis of VLDL triacylglycerol. APOC3 may therefore play an important role in regulating the amount of VLDL production by the liver.

Cellular studies have reported that APOC3 can interfere with TRL and remnant binding to hepatic lipoprotein receptors. APOC3 can abolish APOB- and ApoE-mediated lipoprotein binding to the low-density lipoprotein receptor (LDLR) by either shielding APOB and APOE or altering their conformation. Binding of chylomicrons and VLDL particles to the lipolysis-stimulating receptor (LSR) is also significantly inhibited by APOC3.

Increased APOC3 levels lead to the development of hypertriglyceridemia, i.e. high (hyper-) triglyceride blood levels (-emia). Elevated triglyceride levels are associated with a variety of diseases, including cardiovascular disease, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovarian syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), hypertension, and skin lesions (xanthomas). Extremely high triglyceride levels also increase the risk of acute pancreatitis. Thus, modulating APOC3 metabolism may be an important novel therapeutic approach for managing hypertriglyceridemia and related diseases.

Therefore, there is a need in the art for modulators of APOC3 expression to treat apolipoprotein C3-related disorders, such as hypertriglyceridemia.

The present invention provides iRNA compositions that inhibit or reduce expression of the APOC3 gene. The gene can be in a cell, e.g., in a cell that is in the body of a subject, such as a human.

The present invention also provides methods and therapies for treating subjects having disorders that may benefit from inhibiting or reducing expression of the APOC3 gene, such as apolipoprotein C3-related diseases, such as hypertriglyceridemia, using iRNA compositions that inhibit or reduce expression of the APOC3 gene.

In some embodiments, the present invention provides a double-stranded RNAi agent for inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2;
Substantially all of the nucleotides of at least one strand are modified nucleotides, and the sense strand is conjugated with a ligand attached to the 3' end.

In certain embodiments, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides. In one embodiment, the sense and antisense strands comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sequences listed in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13.

In some embodiments, at least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally fixed nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5- The nucleotide is selected from the group consisting of an anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-methylphosphonate group, a nucleotide containing a 5' phosphate or a 5' phosphate mimic, a nucleotide containing a vinyl phosphate, a nucleotide containing adenosine-glycol nucleic acid (GNA), a nucleotide containing a thymidine-glycol nucleic acid (GNA) S-isomer, a nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide containing 2'-deoxythymidine-3' phosphate, a nucleotide containing 2'-deoxyguanosine-3' phosphate, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

In one embodiment, substantially all of the nucleotides in the sense strand are modified. In another aspect, substantially all of the nucleotides in the antisense strand are modified. In yet another embodiment, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides. In one embodiment, all of the nucleotides in the sense strand are modified nucleotides. In another embodiment, all of the nucleotides in the antisense strand are modified nucleotides. In yet another embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

In one embodiment, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides.

In some embodiments, the invention provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprising a region complementary to a portion of an mRNA encoding APOC3, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III):
Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'
Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5' (III)
[Wherein:
i, j, k, and l are each independently 0 or 1;
p, p', q, and q' are each independently 0 to 6;
each Na and Na' independently represents an oligonucleotide sequence containing 0-25 nucleotides that are either modified or unmodified or a combination thereof, each sequence containing nucleotides of at least two different modifications;
each Nb and Nb' independently represents an oligonucleotide sequence comprising 0-10 nucleotides that are either modified or unmodified or a combination thereof;
each np, np', nq, and nq' (each of which may be present or absent) independently represents an overhanging nucleotide;
XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides;
the modification on Nb is different from the modification on Y, and the modification on Nb' is different from the modification on Y'; and the sense strand is conjugated to at least one ligand.

In a further embodiment, i is 0; j is 0; i is 1; j is 1; i and j are both 0; or i and j are both 1. In another further embodiment, k is 0; l is 0; k is 1; l is 1; k and l are both 0; or k and l are both 1. In another aspect, the YYY motif is present at or near the cleavage site of the sense strand. In yet another aspect, the Y'Y'Y' motif is present at positions 11, 12, and 13 from the 5' end of the antisense strand.

In one embodiment, Y' is 2'-O-methyl or 2'-fluoro.

In some embodiments, formula (III) has formula (IIIa):
Sense: 5'np-Na-YYY-Na-nq3'
Antisense: 3'np'-Na'-Y'Y'Y'-Na'-nq'5' (IIIa)
It is represented by:

In a further aspect, the double-stranded region is 15-30 nucleotide pairs long. In another aspect, the double-stranded region is 17-23 nucleotide pairs long. In another embodiment, the double-stranded region is 17-25 nucleotide pairs long. In yet another embodiment, the double-stranded region is 23-27 nucleotide pairs long. In a further aspect, the double-stranded region is 19-21 nucleotide pairs long. In yet another aspect, the double-stranded region is 21-23 nucleotide pairs long.

In one embodiment, each strand has 15-30 nucleotides. In a further embodiment, each strand has 19-30 nucleotides.

In one embodiment, the modification on the nucleotide is selected from the group consisting of modifications as listed in Tables 5, 9, 10, 11B, 12, 13, and combinations thereof.

In some embodiments, the modifications on the nucleotide are 2'-O-methyl and 2'-fluoro modifications.

In some embodiments, the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. In further embodiments, the ligand is
It is.

In some embodiments, the ligand is attached to the 3' end of the sense strand.

In certain embodiments, the RNAi agent is represented by the following schematic diagram:
wherein X is O or S.

In some embodiments, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In further embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. In another further embodiment, the strand is the antisense strand. In yet another embodiment, the strand is the sense strand.

In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. In a further aspect, this strand is the antisense strand. In another further aspect, this strand is the sense strand.

In certain embodiments, phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand. In one embodiment, this strand is the antisense strand.

In some embodiments, the RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In further embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

In some embodiments, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

In some embodiments, the Y nucleotide contains a 2'-fluoro modification. In further embodiments, the Y' nucleotide contains a 2'-O-methyl modification.

In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

In some embodiments, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, and 12.

In certain embodiments, the present invention also provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region,
the sense strand comprises 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO:13) and the antisense strand comprises 5'-AGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO:14);
substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides;
The sense strand is conjugated with a ligand attached to the 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

In another embodiment, the present invention also provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region,
the sense strand comprises 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-UGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO: 15);
substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides;
The sense strand is conjugated with a ligand attached to the 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

In certain embodiments, the present invention also provides a double-stranded RNAi agent capable of inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region,
the sense strand comprises 5'-GCUUAAAAGGGACAGUAUUCA-3' (SEQ ID NO:659) and the antisense strand comprises 5'-UGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO:670);
substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides;
The sense strand is conjugated with a ligand attached to the 3' end, and the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

In an embodiment, all of the nucleotides in the sense strand are modified nucleotides. In one embodiment, all of the nucleotides in the antisense strand are modified nucleotides. In another embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

In a further embodiment, at least one of the modified nucleotides is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally fixed nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-aryl nucleotide, a 2' ... The nucleotide is selected from the group consisting of an anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-methylphosphonate group, a nucleotide containing a 5' phosphate or a 5' phosphate mimic, a nucleotide containing a vinyl phosphate, a nucleotide containing adenosine-glycol nucleic acid (GNA), a nucleotide containing a thymidine-glycol nucleic acid (GNA) S-isomer, a nucleotide containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, a nucleotide containing 2'-deoxythymidine-3' phosphate, a nucleotide containing 2'-deoxyguanosine-3' phosphate, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

In one embodiment, the RNAi agent comprises 10 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 9 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 8 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 7 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 6 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the RNAi agent comprises 5 or fewer nucleotides that comprise a 2'-fluoro modification. In yet another embodiment, the sense strand comprises 4 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the sense strand comprises 4 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the sense strand comprises 3 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the sense strand comprises 2 or fewer nucleotides that comprise a 2'-fluoro modification. In another aspect, the antisense strand comprises 6 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the antisense strand comprises 5 or fewer nucleotides that comprise a 2'-fluoro modification. In another embodiment, the antisense strand includes no more than four nucleotides that include a 2'-fluoro modification. In another embodiment, the antisense strand includes no more than three nucleotides that include a 2'-fluoro modification. In yet another aspect, the antisense strand includes no more than two nucleotides that include a 2'-fluoro modification.

In one embodiment, the double-stranded RNAi agent of the invention further comprises a 5'-phosphate or a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In another embodiment, the double-stranded RNAi agent further comprises a 5'-phosphate mimic at the 5' nucleotide of the antisense strand. In a specific embodiment, the 5'-phosphate mimic is 5'-vinyl phosphate (5'-VP).

In certain embodiments, the ligand is
It is.

In some embodiments, the RNAi agent is shown in the following schematic diagram:
wherein X is O or S.

In some aspects, the present invention provides a double-stranded RNAi agent comprising an RNAi sequence listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13.

In one embodiment, the RNAi agent has the following sequence:
Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3' (SEQ ID NO: 16)
Antisense: AD-57553 containing 5'asGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsAfsa 3' (SEQ ID NO: 17).

In another embodiment, the RNAi agent has the following sequence:
Sense: 5'GfscsUfuAfaAfaGfGfGfaCfaGfuAfuUfcUfL96 3' (SEQ ID NO: 18)
Antisense: AD-65696 containing 5'VPusGfsaAfuAfcUfgUfcccUfuUfuAfaGfcsasa 3' (SEQ ID NO: 19).

In yet another embodiment, the RNAi agent has the following sequence:
Sense: 5' gscsuuaaAfaGfGfGfacagauucaL96 3' (SEQ ID NO: 20)
Antisense: AD-65703 containing 5'usGfsaauAfcUfGfucccUfuUfuaagcsasa 3' (SEQ ID NO:21).

In yet another embodiment, the RNAi agent has the following sequence:
Sense: 5' gscsuuaaAfaGfGfGfacagauucaL96 3' (SEQ ID NO: 22)
Antisense: AD-65704 containing 5'usGfsaauacugucccUfuuuaagcsasa 3' (SEQ ID NO:23).

In yet another embodiment, the RNAi agent has the following sequence:
Sense: 5' cscscaauAfaAfGfCfuggacaagaaL96 3' (SEQ ID NO: 714)
Antisense: AD-67221 containing 5'usUfscuuGfuCfCfagcuUfuAfuugggsasg 3' (SEQ ID NO:718).

In one embodiment, the RNAi agent has the following sequence:
Sense: 5' gscsuuaaaaGfgGfacagauuca 3' (SEQ ID NO: 738)
Antisense: AD-69535 containing 5'sGfsaauacugucCfcUfuuuaagcsasa 3' (SEQ ID NO:749).

In another embodiment, the RNAi agent has the following sequence:
Sense: 5' gscsuuaaaaGfgGfacagu (Agn) uuca 3' (SEQ ID NO: 744)
Antisense: AD-69541 containing 5'usGfsaauacugucCfcUfuuuaagcsasa 3' (sequence number 755).

In certain embodiments, the present invention also provides a composition comprising a modified antisense polynucleotide agent capable of inhibiting expression of APOC3 in a cell and comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, the polynucleotide being about 14 to about 30 nucleotides in length.

In some aspects, the present invention also provides vectors containing the double-stranded RNAi agents as described herein. In other aspects, the present invention also provides cells containing the double-stranded RNAi agents as described herein.

In some embodiments, the present invention relates to a pharmaceutical composition comprising a double-stranded RNAi agent, or a composition comprising a modified antisense polynucleotide agent, or a vector, as described herein.

In certain embodiments, the double-stranded RNAi agent is present in a non-buffered solution. In further embodiments, the non-buffered solution is saline or water. In other embodiments, the double-stranded RNAi agent is present in a buffered solution. In further embodiments, the buffered solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a specific embodiment, the buffered solution is phosphate buffered saline (PBS).

In one embodiment, the present invention also provides a method of inhibiting apolipoprotein C3 (APOC3) expression in a cell, the method comprising:
(a) contacting a cell with a composition, vector, or pharmaceutical composition comprising a double-stranded RNAi agent, or a modified antisense polynucleotide agent, as described herein;
(b) maintaining the cells resulting from step (a) for a period of time sufficient to effect degradation of mRNA transcripts of the APOC3 gene, thereby inhibiting expression of the APOC3 gene in the cells.

In one aspect, the cell is in a subject. In a further aspect, the subject is a human or rabbit. In one embodiment, the subject is suffering from an APOC3-associated disease.

In some embodiments, APOC3 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

In some aspects, the present invention provides a method of treating a subject having an apolipoprotein C3 (APOC3)-associated disease, the method comprising administering to the subject a therapeutically effective amount of a composition, vector, or pharmaceutical composition comprising a double-stranded RNAi agent, or a modified antisense polynucleotide agent, as described herein, thereby treating the subject.

In one embodiment, the APOC3-related disorder is hypertriglyceridemia. In another embodiment, the APOC3-related disorder is selected from the group consisting of nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance), hypertension, atherosclerosis, and pancreatitis.

In some embodiments, the double-stranded RNAi agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg. In further embodiments, the double-stranded RNAi agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In another embodiment, the double-stranded RNAi agent is administered at a dose of about 3 mg/kg. In yet another embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg/kg.

In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In another embodiment, the double-stranded RNAi agent is administered intravenously. In another embodiment, the double-stranded RNAi agent is administered intramuscularly.

In some embodiments, the RNAi agent is administered in two or more doses. In further embodiments, the RNAi agent is administered at intervals selected from the group consisting of about once every 12 hours, about once every 24 hours, about once every 48 hours, about once every 72 hours, and about once every 96 hours.

In certain embodiments, the method of the present invention further comprises administering to the subject an additional therapeutic agent. In further embodiments, the additional therapeutic agent is selected from the group consisting of HMG-CoA reductase inhibitors, fibrates, bile acid sequestrants, niacin, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTTP) inhibitors, cholesterol modulators, bile acid modulators, peroxisome proliferator-activated receptor (PPAR) agonists, gene-based therapies, combined vascular protectors, glycoprotein Ilb/IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors, squalene synthase inhibitors, monocyte chemotactic protein (MCP)-I inhibitors, or fish oil.

1 is a bar graph showing the relative amount of APOC3 mRNA in Hep3B cells following treatment with a single dose of 0.1 nM or 10 mM of the indicated iRNA of the invention. 1 is a bar graph showing the relative amounts of APOC3 mRNA measured on day 5 in wild-type mice treated with 3, 10 and 30 mg/kg doses of GalNac-conjugated AD-57558. 1 is a bar graph showing measurements of APOC3 mRNA levels measured in individual APOC3-AAV mice injected with AD-57553, AD-57547, and AD-58924. 1 is a bar graph showing group means of APOC3 mRNA levels measured in APOC3-AAV mice injected with AD-57553, AD-57547, and AD-58924. 1 is a bar graph showing the relative amount of APOC3 mRNA measured in APOC3-AAV mice pre-injected with 10 11 hAPOC3 AAV genome copies followed by injection of 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg doses of AD-57553. 1 is a bar graph showing group means of relative amounts of APOC3 mRNA measured in APOC3-AAV mice pre-injected with 10 11 hAPOC3 AAV genome copies followed by injection of 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg doses of AD-57553. Graph showing the 20 day time course of serum APOC3 protein measured in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 3 mg/kg dose of the indicated iRNA of the invention. Graph showing the 30 day time course of serum APOC3 protein measured in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 3 mg/kg dose of the indicated iRNA of the invention. 1 is a bar graph showing the amount of serum APOC3 protein measured on day 10 in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 3 mg/kg dose of the indicated iRNA of the invention. 1 is a bar graph showing the amount of serum APOC3 protein measured on day 20 in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 3 mg/kg dose of the indicated iRNA of the invention. 1 is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 3 mg/kg dose of the indicated iRNA of the invention. FIG. 1 is a schematic diagram showing the dosing schedule Q2W×4 used in multiple dose studies with AD-57553, AD-65696, AD-65699, AD-65703, and AD-65704. Figure 12A is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 1011 hAPOC3 AAV genome copies followed by four 0.3 mg/kg doses of the indicated iRNA of the invention administered according to the dosing schedule shown in Figure 11. Figure 12B is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 1011 hAPOC3 AAV genome copies followed by four 1 mg/kg doses of the indicated iRNA of the invention administered according to the dosing schedule shown in Figure 11. Figure 12C is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 1011 hAPOC3 AAV genome copies followed by four 3 mg/kg doses of the indicated iRNA of the invention administered according to the dosing schedule shown in Figure 11. 1 is a bar graph showing the relative amount of APOC3 protein measured on day 14 in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a single dose of 0.3 mg/kg, 1 mg/kg and 3 mg/kg AD-65704. FIG. 12 is a bar graph showing the relative amount of APOC3 protein measured 20 days after the final dose in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by multiple doses of 0.3 mg/kg, 1 mg/kg and 3 mg/kg AD-65704 administered according to the dosing schedule shown in FIG. 1 is a time course showing the amount of APOC3 protein measured in APOC3-AAV mice injected with 10 11 hAPOC3 AAV genome copies followed by a 1 mg/kg dose of the indicated iRNA of the invention. Figure 16A is a bar graph showing the relative amount of APOC3 protein measured on day 10 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a single 1 mg/kg dose of the indicated iRNA of the invention. Figure 16B is a bar graph showing the relative amount of APOC3 protein measured on day 24 in APOC3-AAV mice injected with 10 hAPOC3 AAV genome copies followed by a single 1 mg/kg dose of the indicated iRNA of the invention. Figure 17A is a bar graph showing the relative amounts of serum APOC3 protein measured on day 14 in APOC3-AAV mice injected with 1011 hAPOC3 AAV genome copies followed by a single 1 mg/kg dose of the indicated iRNA. Figure 17B is a graph showing the amounts of serum APOC3 protein measured on days 0, 14, 28, and 42 relative to pre-dose levels in APOC3-AAV mice injected with 1011 hAPOC3 AAV genome copies followed by a single 1 mg/kg dose of the indicated iRNA. Figure 18A is a graph showing serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, 36, 43, 57, 64, and 71 in cynomolgus monkeys after a single 1 mg/kg weekly dose of AD-65704 (QWx8) for 8 weeks compared to pre-dose on day -7. Figure 18B is a graph showing serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, and 36 in cynomolgus monkeys after a single 1 mg/kg dose of AD-65704 compared to pre-dose on day -7. FIG. 18C is a graph showing liver APOC3 mRNA on day 64 in cynomolgus monkeys after a single 1 mg/kg weekly dose of AD-65704 (q1w×5) for 5 weeks compared to pre-dosing on day −7, and liver APOC3 mRNA on day 12 in cynomolgus monkeys after a single 1 mg/kg dose of AD-65704 compared to pre-dosing on day −7. Figure 19A is a graph showing serum APOC3 protein measured on days 1, 8, 11, 15, 22, 29, and 36 in cynomolgus monkeys after a single 1 mg/kg dose of the indicated iRNA compared to pre-dosing on day -7. Figure 19B is a bar graph showing liver APOC3 mRNA measured on day 12 in cynomolgus monkeys after a single 1 mg/kg dose of the indicated iRNA compared to pre-dosing on day -7. Figure 20A is a graph showing serum APOC3 mRNA measured on days 1, 8, 11, 15, 22, 29, 36, 43, 50, 57, 64, and 71 in cynomolgus monkeys following administration of a single 1 mg/kg dose of the indicated iRNA followed by a single subcutaneous 3 mg/kg dose of the same agent on day 36, compared to pre-dosing on day -7. Figure 20B is a bar graph showing liver APOC3 mRNA measured on day 12 in cynomolgus monkeys following a single 1 mg/kg dose of the indicated iRNA on day 1 followed by a single 3 mg/kg dose of the same iRNA agent on day 36, compared to pre-dosing on day -7.

The present invention provides iRNA agents, e.g., double-stranded iRNA agents, and compositions that reduce or inhibit expression of the APOC3 gene. The gene can be in a cell, e.g., in a cell that is in the body of a subject, such as a human.

The present invention also provides methods of treating a subject having a disorder that would benefit from inhibiting or reducing expression of APOC3, e.g., an apolipoprotein C3-related disease or disorder, such as hypertriglyceridemia, using an iRNA composition that inhibits or reduces expression of the APOC3 gene.

The iRNA of the present invention is about 30 nucleotides in length or less, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19- The iRNAs comprise an RNA strand (antisense strand) having a region of 24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the APOC3 gene. These iRNAs can be used to target and degrade the mRNA of the APOC3 gene in cells. In particular, extremely low dosages of the iRNAs of the present invention can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of the APOC3 gene. Using in vitro and in vivo assays, the inventors have demonstrated that iRNAs targeting the APOC3 gene can mediate RNAi, resulting in significant inhibition of APOC3 gene expression and reduced APOC3 protein levels. The inventors have also demonstrated that iRNAs targeting the APOC3 gene can reduce symptoms associated with apolipoprotein C3-related disorders, such as reducing triglyceride levels. Thus, methods and compositions including these iRNAs are useful for treating subjects with apolipoprotein C3-related disorders, such as hypertriglyceridemia.

The detailed description below discloses how to make and use compositions containing iRNA to inhibit expression of the APOC3 gene, as well as compositions, uses, and methods for treating subjects with diseases and disorders that may benefit from inhibition and/or reduction of APOC3 expression.

I. Definitions In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values for a parameter is listed, it is intended that values and ranges intermediate to the listed values are also part of the present invention.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or two or more elements, e.g., a plurality of elements.

The term "including" is used herein to mean, and is used interchangeably with, the term "including, but not limited to."

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless the context clearly indicates otherwise.

As used herein, the term "APOC3" refers to the well-known gene encoding apolipoprotein C3 and its protein product, also known in the art as HALP2 or APOCIII.

The term "APOC3" includes human APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank Accession No. GI:4557322 (NM_000040.1; SEQ ID NO:1)); Macaca fascicularis APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank Accession No. GI:544489959 (XM_05579730.1; SEQ ID NO:3)); Macaca mulatta APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank Accession No. GI:297269260 (XM_001090312.2; SEQ ID NO:5)); mouse (Mus musculus) APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank Accession No. GI:297269260 (XM_001090312.2; SEQ ID NO:5)); musculus) APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank Accession No. GI: 577019555 (NM_023114.4, SEQ ID NO: 7)); rat (Rattus norvegicus) APOC3 (for its amino acid and complete coding sequence, see, for example, GenBank Accession No. GI: 402534545 (NM_012501.2, SEQ ID NO: 9)); and rabbit (Oryctolagus cuniculus), GenBank Accession No. GI: 655601498 (XM_002708371.2, SEQ ID NO: 11).

Additional examples of APOC3 mRNA sequences are readily available in publicly available databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

The term "APOC3," as used herein, also refers to naturally occurring DNA sequence variations of the APOC3 gene, such as single nucleotide polymorphisms (SNPs) of the APOC3 gene. Exemplary SNPs of the APOC3 DNA sequence may be found in the dbSNP database available at www.ncbi.nlm.nih.gov/projects/SNP/. Non-limiting examples of sequence variations within the APOC3 gene include, for example, the two variations rs2854116 and rs2854117 described in Petersen, K. F. et al., (2010), N. Engl. J. Med. 362(12):1082-1089, the entire contents of which are incorporated herein by reference.

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the APOC3 gene, including the mRNA that is the RNA processing product of the primary transcript. In one embodiment, the target portion of the sequence is at least sufficiently long to serve as a substrate for iRNA-directed cleavage at or near the portion of the nucleotide sequence of an mRNA molecule formed during transcription of the APOC3 gene.

The target sequence may be about 9-36 nucleotides in length, such as about 15-30 nucleotides in length. For example, the target sequence may be 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-2 The length may be about 15-30 nucleotides, such as 6, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

As used herein, the term "strand containing a sequence" refers to an oligonucleotide that contains a strand of nucleotides described by a referenced sequence, using standard nucleotide nomenclature.

"G", "C", "A", "T" and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 3). Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. As a non-limiting example, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine can be replaced, for example, with a nucleotide containing inosine in the nucleotide sequence of a dsRNA featured in the present invention. In another example, adenine and cytosine can be substituted with guanine and uracil, respectively, anywhere in the oligonucleotide to form a G-U wobble base pair with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods featured herein.

The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" are used interchangeably herein and refer to an agent that contains RNA, as defined herein, and mediates targeted cleavage of an RNA transcript through the RNA-induced silencing complex (RISC) pathway. iRNA induces sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA modulates, e.g., inhibits, APOC3 expression in a cell, e.g., a cell in a subject, e.g., a mammalian subject.

In one embodiment, the RNAi agent of the invention comprises a single stranded RNA that interacts with a target RNA sequence, e.g., an APOC3 target mRNA sequence, and induces cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into a cell is degraded into double stranded short interfering RNAs (siRNAs) containing a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). When Dicer, a ribonuclease III-like enzyme, processes these dsRNAs, they become 19-23 base pair short interfering RNAs with unique 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present invention relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is generated in a cell and promotes the formation of a RISC complex to cause silencing of a target gene, i.e., the APOC3 gene. Thus, the term "siRNA" is also used herein to refer to RNAi as described above.

In another embodiment, the RNAi agent may be a single stranded RNA that is introduced into a cell or organism to inhibit the target mRNA. The single stranded RNAi agent binds to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. Single stranded siRNAs are generally 15-30 nucleotides and are chemically modified. Design and testing of single stranded RNAs are described in U.S. Pat. No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated by reference herein. Any of the antisense nucleotide sequences described herein may be used as single stranded siRNA as described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, the "iRNA" used in the compositions, uses, and methods of the invention is double-stranded RNA, referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," "RNAi agent," "RNAi," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to a target RNA, i.e., the APOC3 gene. In some embodiments of the invention, the double-stranded RNA (dsRNA) causes degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

Generally, the majority of the nucleotides in each strand of a dsRNA molecule are ribonucleotides, but each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and/or modified nucleotides, as described in detail herein. In addition, as used herein, an "RNAi agent" may include a ribonucleotide having a chemical modification; an RNAi agent may include substantial modifications to multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and/or a modified nucleobase. Thus, the term modified nucleotide includes the substitution, addition, or removal of, for example, a functional group or atom to an internucleoside linkage, sugar moiety, or nucleobase. Modifications suitable for use in agents of the invention include any type of modification disclosed herein or known in the art. Any such modification is included in the "RNAi agent" for purposes of this specification and claims when used in siRNA type molecules.

The double-stranded region may be of any length that allows for specific degradation of the desired target RNA through the RISC pathway, and may range from about 9 to 36 base pairs in length, for example, about 15 to 30 base pairs in length, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example, about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 1 9-22, 19-21, Such as 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

The two strands forming the double-stranded structure may be different parts of a larger RNA molecule, or they may be separate RNA molecules. When the two strands are part of one larger molecule, and thus are joined by an uninterrupted stretch of nucleotides between the 3' end of one strand and the 5' end of each other strand forming the double-stranded structure, the joined RNA strands are referred to as "hairpin loops." A hairpin loop may contain at least one unpaired nucleotide; in some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.

When the two substantially complementary strands of a dsRNA are constituted by another RNA molecule, these molecules may, but need not, be covalently linked. When the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of each of the other strands, the linking structure is called a "linker". The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the double strand. In addition to the double-stranded structure, the RNAi may include one or more nucleotide overhangs.

In one embodiment, the RNAi agents of the invention are dsRNAs, each strand of which contains 20-30 nucleotides that interact with a target RNA sequence, e.g., an APOC3 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 base pair small interfering RNAs with unique 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, e.g., a dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleotides. A nucleotide overhang can include or consist of nucleotide/nucleoside analogs, including deoxyribonucleotides/nucleosides. An overhang can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the overhanging nucleotides can be present on the 5' end, the 3' end, or both ends of either the antisense or sense strand of the dsRNA.

In one embodiment, the antisense strand of the dsRNA has 1-10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and/or 5' end. In one embodiment, the sense strand of the dsRNA has 1-10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and/or 5' end. In another embodiment, one or more nucleotides in the overhang are substituted with a thiophosphate nucleoside.

"Blunt ended" or "blunt ended" means that there are no unpaired nucleotides, i.e., no nucleotide overhangs, at that end of a double-stranded RNAi agent. A "blunt ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at either end of the molecule. RNAi agents of the present invention include RNAi agents that have a nucleotide overhang at one end (i.e., an agent with one overhang and one blunt end) or that have nucleotide overhangs at both ends.

The term "antisense strand" or "guide strand" refers to an iRNA strand, such as a dsRNA, that includes a region that is substantially complementary to a target sequence, such as, for example, an APOC3 mRNA. As used herein, the term "region complementary" refers to a region on the antisense strand that is substantially complementary to a sequence, such as, for example, a target sequence, such as, for example, an APOC3 nucleotide sequence as defined herein. When the region of complementarity is not perfectly complementary to the target sequence, there can be mismatches in internal or terminal regions of the molecule. In general, mismatches that are most tolerated are in the terminal regions, such as, for example, within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' ends of the iRNA.

The term "sense strand" or "passenger strand," as used herein, refers to an iRNA strand that includes a region that is substantially complementary to a region of the antisense strand, as defined herein.

As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site where cleavage occurs on the target. In some embodiments, the cleavage region includes 3 bases on either side of and immediately adjacent to the cleavage site. In some embodiments, the cleavage region includes 2 bases on either side of and immediately adjacent to the cleavage site. In some embodiments, the cleavage site is specifically located at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage region includes nucleotides 11, 12, and 13.

As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions to produce a double-stranded structure, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES at pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may be applied. Those skilled in the art can determine the optimal set of conditions for testing the complementarity of two sequences according to the end use of the hybridized nucleotides.

Complementary sequences in an iRNA, such as in a dsRNA described herein, include base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to one another. However, when a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3 or 2 mismatched base pairs upon hybridization of a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most appropriate for their end use, such as, for example, inhibition of gene expression through the RISC pathway. However, if the two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered as mismatches for purposes of determining complementarity. For example, a dsRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide contains a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, is still referred to as "perfectly complementary" for purposes described herein.

"Complementary" sequences, as used herein, also include or may be formed entirely from non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, so long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs.

As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to base matching between the sense and antisense strands of a dsRNA or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context in which they are used.

As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of a target mRNA (e.g., an mRNA encoding APOC3). For example, a polynucleotide is complementary to at least a portion of an APOC3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding APOC3.

Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target APOC3 sequence. In other embodiments, the sense strand polynucleotides and/or antisense polynucleotides disclosed herein are substantially complementary to the target APOC3 sequence, and include a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over the entire length of the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-12, or a fragment of any one of SEQ ID NOs: 1-12.

In one embodiment, the RNAi agent of the present invention comprises a sense strand substantially complementary to an antisense polynucleotide complementary to a target APOC3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to an equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1-12, or a fragment of any one of SEQ ID NOs: 1-12. In another embodiment, the RNAi agent of the present invention comprises an antisense strand substantially complementary to a target APOC3 sequence, and comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to the equivalent region of any one of SEQ ID NOs: 1-12, or a fragment of any one of SEQ ID NOs: 1-12.

Generally, the majority of the nucleotides in each strand are ribonucleotides, although, as described in detail herein, one or both strands may also include one or more non-ribonucleotides, such as, for example, deoxyribonucleotides and/or modified nucleotides. Additionally, "iRNA" includes ribonucleotides that have chemical modifications. Such modifications include all types of modifications disclosed herein or known in the art. For purposes of this specification and claims, all such modifications are encompassed by "iRNA" as used in iRNA molecules.

In one aspect of the invention, the agent used in the methods and compositions of the invention is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA by an antisense inhibition mechanism. The single-stranded antisense nucleic acid molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing with the mRNA and physically interfering with the translation machinery (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense nucleic acid molecule can be about 15 to about 30 nucleotides in length and can have a sequence complementary to the target sequence. For example, the single-stranded antisense nucleic acid molecule can include a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

As used herein, a "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., monkey, chimpanzee, etc.), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder, or condition that would benefit from reduced APOC3 expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced APOC3 expression; a human having a disease, disorder, or condition that would benefit from reduced APOC3 expression; and/or a human being treated for a disease, disorder, or condition that would benefit from reduced APOC3 expression as described herein.

As used herein, the term "treat" or "treatment" refers to a beneficial or desired result, including but not limited to, the alleviation or amelioration of one or more symptoms associated with undesirable or excessive APOC3 expression, such as hypertriglyceridemia (or high triglyceride levels). Such symptoms may include, for example, skin symptoms (e.g., eruptive xanthomas); eye abnormalities (e.g., lipemia retinalis); hepatosplenomegaly (enlargement of the liver and spleen); neurological symptoms; or abdominal pain attacks, which may be mild episodes of pancreatitis. Other symptoms associated with undesirable or excessive APOC3 expression may also include any disease, disorder, or condition that may be caused by, associated with, or result from hypertriglyceridemia, such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovarian syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term "lower" in the context of a subject's APOC3 levels or disease markers or symptoms refers to a statistically significant reduction in such levels. This reduction may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, preferably down to a level accepted as being within the normal range for individuals without such disorder.

As used herein, "prevention" or "preventing," when used in connection with a disease, disorder, or condition that can be treated or ameliorated by reducing expression of the APOC3 gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, e.g., symptoms of undesirable or excessive APOC3 expression, such as hypertriglyceridemia. For example, the likelihood of developing hypertriglyceridemia is reduced, e.g., when an individual having one or more risk factors for hypertriglyceridemia does not develop hypertriglyceridemia or develops hypertriglyceridemia with reduced severity compared to a population having the same risk factors and not receiving treatment as described herein. Not developing a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically accepted scale for the disease or disorder in question), or a delay in symptoms (e.g., by days, weeks, months, or years) is considered effective prevention.

As used herein, the term "apolipoprotein C3-related disease" or "APOC3-related disease" is a disease, disorder, or condition caused by or associated with unwanted or excessive APOC3 expression. The term "APOC3-related disease" includes diseases, disorders, or conditions that may be treated or ameliorated by reducing APOC3 expression. The term "APOC3-related disease" includes hypertriglyceridemia, or high triglyceride levels.

Triglyceride levels in serum of subjects, e.g., human subjects, that may be indicative of hypertriglyceridemia are described in Oh, R. C. et al., (2007) American Family Physician, 75(9):1366-1371. Specifically, hypertriglyceridemia may be associated with "borderline high serum triglyceride levels" (i.e., 150-199 mg/dL or 1.70-2.25 mmol/L); "high serum triglyceride levels" (i.e., 200-499 mg/dL or 2.26-5.64 mmol/L); or "very high triglyceride levels" (i.e., 500 mg/dL or higher (or 5.65 mmol/L or higher).

In one embodiment, the APOC3-related disease is primary hypertriglyceridemia. "Primary triglyceridemia" results from environmental or genetic causes (e.g., as a result of an obvious underlying disease). Exemplary diseases characterized as primary hypertriglyceridemia include, but are not limited to, familial chylomicroemia (hyperlipoproteinemia type I), primary mixed hyperlipidemia (type 5), familial hypertriglyceridemia (hyperlipoproteinemia type 4), familial combined hyperlipoproteinemia (type 2B), and familial dysbetalipoproteinemia (hyperlipoproteinemia type 3).

In another embodiment, the APOC3-related disorder is secondary hypertriglyceridemia. "Secondary hypertriglyceridemia" is caused by or associated with other underlying disorders and conditions. Such disorders and/or conditions include, for example, obesity, metabolic syndrome, diabetes, fatty liver, alcohol consumption, kidney disease, pregnancy, nonalcoholic fatty liver disease, hypothyroidism, paraproteinemia (such as hypergammaglobulinemia in macroglobulinemia, myeloma, lymphoma and lymphocytic leukemia), autoimmune disorders (such as systemic lupus erythematosus), and medications (such as antiretroviral drugs including ritonavir and lopinavir, and antipsychotic therapy including clozapine and olanzapine) (see G. Yuan et al., (2007) Canadian Medical Association Journal, 176(8):1113-1120).

Any disorder that can cause hypertriglyceridemia (e.g., secondary hypertriglyceridemia) or that can be a result of hypertriglyceridemia (e.g., primary or secondary hypertriglyceridemia) is encompassed by the term "APOC3-associated disease." Non-limiting examples of APOC3-associated diseases include metabolic disorders, e.g., nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovarian syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance); hypertension; cardiovascular disorders, e.g., atherosclerosis; and pancreatitis, e.g., acute pancreatitis.

II. iRNA of the present invention
The present invention provides an iRNA that inhibits the expression of APOC3 gene.In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of APOC3 gene in a cell, such as a cell in a mammalian body, such as a human, subject with APOC3-related disease, such as hypertriglyceridemia.The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed by the expression of APOC3 gene.The complementary region is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the APOC3 gene, the iRNA inhibits expression of the APOC3 gene (e.g., a human, primate, non-primate, or avian APOC3 gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as by immunofluorescence analysis, for example using Western blotting or flow cytometry.

dsRNA contains two complementary RNA strands that hybridize to form a double-stranded structure under conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary, and generally completely complementary, to a target sequence. The target sequence may be derived from the sequence of an mRNA formed during expression of the APOC3 gene. The other strand (the sense strand) contains a region complementary to the antisense strand such that, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere herein and known in the art, the complementary sequences of the dsRNA may also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

In general, the double-stranded structure is, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27 , 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

Similarly, the complementary region of the target sequence may be, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-2 7, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the recited ranges and lengths are also intended to be part of the invention.

In some embodiments, the dsRNA is about 15 to about 20 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to act as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides in length may act as substrates for Dicer. As one of skill in the art will recognize, the target region of an RNA that is targeted for cleavage is most often a portion of a larger RNA molecule, which is often an mRNA molecule. Where applicable, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage through the RISC pathway).

Those skilled in the art will appreciate that, for example, about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-3 3, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15 ~25, 15~24, 15~23, 15~22, 15~21, 15~20, 15~1 9, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24 , 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20 It will also be appreciated that the double-stranded region, such as a double-stranded region of about 9-36 base pairs, such as 28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, is the primary functional portion of the dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs, within which it is processed, for example, into a functional duplex of 15-30 base pairs that targets a desired RNA for cleavage, is a dsRNA. Thus, one of skill in the art will appreciate that in one embodiment, a miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting APOC3 expression is not generated in a target cell by cleavage of a larger dsRNA.

The dsRNAs described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs, including deoxyribonucleotides/nucleosides. The overhangs may be on the sense strand, the antisense strand, or any combination thereof. Additionally, the overhanging nucleotides may be present on the 5' end, the 3' end, or both ends of either the antisense or sense strand of the dsRNA.

dsRNA can be synthesized by standard methods known in the art, for example using an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

The iRNA compounds of the invention may be prepared using a two-step process. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound may be prepared using solution phase or solid phase organic or both. Organic synthesis offers the advantage that oligonucleotide strands containing non-natural or modified nucleotides may be easily prepared. The single-stranded oligonucleotides of the invention may be prepared using solution phase or solid phase organic synthesis or both.

In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand and the corresponding antisense strand are each selected from the group of sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. In this embodiment, one of these two sequences is complementary to the other of these two sequences, and one of these sequences is substantially complementary to the sequence of the mRNA resulting from expression of the APOC3 gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. In one embodiment, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.

Although some of the sequences in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 are described as modified and/or conjugated sequences, it will be understood that the RNA of the iRNA of the present invention, e.g., the dsRNA of the present invention, can include any one of the sequences shown in Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 that is unmodified, unconjugated, and/or modified and/or conjugated differently than those described therein.

Those skilled in the art are well aware that dsRNAs having a duplex structure of about 20-23 base pairs, such as 21 base pairs, have been advocated as being particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective. (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-mentioned embodiments, due to the nature of the oligonucleotide sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13, the dsRNAs described herein may include at least one strand of a minimum length of 21 nucleotides. It may be reasonably expected that shorter duplexes having one of the sequences of any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13, which are missing only a few nucleotides at one or both ends, may be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides derived from one of the sequences of any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13, and having an ability to inhibit APOC3 gene expression that differs from a dsRNA containing the full-length sequence by about 5, 10, 15, 20, 25, or 30% or less, are intended to be within the scope of the present invention.

Additionally, the RNAs provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 identify sites in the APOC3 transcript that are susceptible to RISC-mediated cleavage. The invention therefore further features iRNAs that target within one of these sequences. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that specific site. Such iRNAs generally include about 15 contiguous nucleotides from one of the sequences provided in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 linked to additional nucleotide sequences from regions adjacent to the selected sequence in the APOC3 gene.

Target sequences are generally about 15-30 nucleotides in length, although there is wide variability in the suitability of specific sequences within this range to induce cleavage of any given target RNA. While the various software packages and guidelines presented herein provide guidance for identifying optimal target sequences for any given gene target, an empirical approach can also be taken in which a "window" or "mask" of a given size (21 nucleotides as a non-limiting example) is placed, either actually or figuratively (e.g., including by computer simulation), in the target RNA sequence to identify sequences of a size range that can serve as target sequences. By successively moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing to identify optimally functioning sequences (using assays described herein or known in the art), can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, while the sequences identified in, for example, any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12 and 13 represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by successively "walking the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equivalent or better inhibitory properties.

It is contemplated that further optimization of any sequence identified in, for example, any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13 may be achieved by systematically adding or removing nucleotides to create longer or shorter sequences and testing these created sequences by walking from that position through a window of size longer or shorter than the target RNA. Again, combining this approach of creating new target candidates with testing the efficacy of iRNAs based on these target sequences in inhibition assays known in the art and/or described herein may result in further improvements in inhibition efficiency. Still further, such optimized sequences may be adjusted by, for example, introducing modified nucleotides described herein or known in the art, adding or modifying overhangs, or other modifications known in the art and/or discussed herein to further optimize the molecule as an expression inhibitor (e.g., increased serum stability or circulating half-life, increased thermostability, enhanced transmembrane delivery, targeting specific locations or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes, etc.).

The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain no more than three mismatches. When the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the extent of the mismatch is not located in the center of the complementary region. When the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the mismatch is limited to within the last 5 nucleotides from either the 5' or 3' end of the complementary region. For example, in a 23 nucleotide iRNA agent strand complementary to an APOC3 gene region, the RNA strand generally does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an iRNA containing mismatches with the target sequence is effective in inhibiting expression of the APOC3 gene. Consideration of the effectiveness of iRNAs with mismatches in inhibiting expression of the APOC3 gene is important, especially when a particular complementary region of the APOC3 gene is known to have polymorphic sequence variation within the population.

III. Modified iRNAs of the Invention
In one embodiment, the RNA of the iRNA of the invention, e.g., dsRNA, is unmodified and does not contain chemical modifications and/or linkages, e.g., known in the art and described herein. In another embodiment, the RNA of the iRNA of the invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the invention, substantially all of the nucleotides of the iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of the iRNA of the invention are modified nucleotides. An iRNA of the invention in which "substantially all of the nucleotides are modified" is mostly, but not all, modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

In some aspects of the invention, substantially all of the nucleotides of the iRNA of the invention are modified, and the iRNA agent includes 10 or fewer nucleotides that include a 2'-fluoro modification (e.g., 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). For example, in some embodiments, the sense strand includes 4 or fewer nucleotides that include a 2'-fluoro modification (e.g., 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other embodiments, the antisense strand includes 6 or fewer nucleotides that include a 2'-fluoro modification (e.g., 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications). In other aspects of the invention, all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent includes 10 or fewer nucleotides that include 2'-fluoro modifications (e.g., 9 or fewer 2'-fluoro modifications, 8 or fewer 2'-fluoro modifications, 7 or fewer 2'-fluoro modifications, 6 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 5 or fewer 2'-fluoro modifications, 4 or fewer 2'-fluoro modifications, 3 or fewer 2'-fluoro modifications, or 2 or fewer 2'-fluoro modifications).

The nucleic acids featured in the present invention may be synthesized and/or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. For example, modifications include terminal modifications, such as 5'-end modifications (phosphorylation, conjugated linkage, inverted linkage) or 3'-end modifications (conjugated linkage, DNA nucleotide, inverted linkage, etc.); base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; backbone modifications, including modifications or replacement of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones or RNAs that do not contain natural internucleoside linkages. RNAs with modified backbones include, in particular, those that do not have a phosphorus atom in the backbone. For purposes herein, and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone are also considered to be oligonucleosides. In some embodiments, modified iRNAs have a phosphorus atom in their internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and boranophosphates with reversed polarity, where adjacent nucleoside unit pairs are linked 3'-5' to 5'-3', or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

Representative U.S. patents which teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; and 5,286,711, each of which is hereby incorporated by reference in its entirety. No. 17; U.S. Pat. No. 5,321,131; U.S. Pat. No. 5,399,676; U.S. Pat. No. 5,405,939; U.S. Pat. No. 5,453,496; U.S. Pat. No. 5,455,233; U.S. Pat. No. 5,466,677; U.S. Pat. No. 5,476,925; U.S. Pat. No. 5,519,126; U.S. Pat. No. 5,536,821; U.S. Pat. No. 5,541,316; U.S. Pat. No. 5,550,111; U.S. Pat. No. 5,563,253; U.S. Pat. No. 5,571 ,799; U.S. Pat. No. 5,587,361; U.S. Pat. No. 5,625,050; U.S. Pat. No. 6,028,188; U.S. Pat. No. 6,124,445; U.S. Pat. No. 6,160,109; U.S. Pat. No. 6,169,170; U.S. Pat. No. 6,172,209; U.S. Pat. No. 6,239,265; U.S. Pat. No. 6,277,603; U.S. Pat. No. 6,326,199; U.S. Pat. No. 6,346,614; U.S. Pat. No. 6,444,423; U.S. Pat. 531,590; U.S. Pat. No. 6,534,639; U.S. Pat. No. 6,608,035; U.S. Pat. No. 6,683,167; U.S. Pat. No. 6,858,715; U.S. Pat. No. 6,867,294; U.S. Pat. No. 6,878,805; U.S. Pat. No. 7,015,315; U.S. Pat. No. 7,041,816; U.S. Pat. No. 7,273,933; U.S. Pat. No. 7,321,029; and U.S. Pat. No. RE39464, but are not limited thereto.

Modified RNA backbones that do not contain phosphorus atoms therein have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; those with amide backbones; and others with mixed N, O, S, and CH2 components.

Representative U.S. patents which teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; and 5,470,967, each of which is hereby incorporated by reference in its entirety. No. 5,489,677; U.S. Pat. No. 5,541,307; U.S. Pat. No. 5,561,225; U.S. Pat. No. 5,596,086; U.S. Pat. No. 5,602,240; U.S. Pat. No. 5,608,046; U.S. Pat. No. 5,610,289; U.S. Pat. No. 5,618,704; U.S. Pat. No. 5,623,070; U.S. Pat. No. 5,663,312; U.S. Pat. No. 5,633,360; U.S. Pat. No. 5,677,437; and U.S. Pat. No. 5,677,439.

In another embodiment, suitable RNA mimics are contemplated for use in iRNA, in which both the sugar and the internucleoside linkages, i.e., the backbone of the nucleotide units, are replaced with new groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are linked directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the contents of each of which are incorporated herein by reference in their entirety. Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the present invention include RNAs with phosphorothioate backbones, and oligonucleosides with heteroatom backbones that are --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as methylene (methylimino) or MMI backbones], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [natural phosphodiester backbones are represented as --O--P--O--CH2--] of the aforementioned U.S. Pat. No. 5,489,677, and with amide backbones of the aforementioned U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of U.S. Pat. No. 5,034,506, supra.

Modified RNAs may also contain one or more substituted sugar moieties. For example, iRNAs, such as dsRNAs, featured herein may contain one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl at the 2' position, where the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl, or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In another embodiment, the dsRNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group that improves the pharmacokinetic properties of an iRNA, or a group that improves the pharmacodynamic properties of an iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described in the Examples herein below.

Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, specifically at the 3' position of the sugar on the 3' terminal nucleotide, or in 2'-5' linked dsRNAs, and at the 5' position of the 5' terminal nucleotide. An iRNA may also have a sugar mimic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative United States patents which teach the preparation of the above modified sugar structures include, certain of which are commonly owned with the present application, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; and 5,519,135. Nos. 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, the contents of each of which are hereby incorporated by reference in their entirety.

iRNAs may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxy-thymine (dT), 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil); Other synthetic and natural nucleobases include 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine, and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Y. S. , Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. , Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), making them an exemplary base substitution, even more so when combined with 2'-O-methoxyethyl sugar modifications.

Representative U.S. patents which teach the preparation of the above-mentioned specific modified nucleobases as well as other modified nucleobases include U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; and 5,587,469, each of which is hereby incorporated by reference in its entirety. Examples of such applications include, but are not limited to, U.S. Patent Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088.

The RNA of the iRNA may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, the agent of the invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide that includes a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-terminal structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include, without limitation, nucleosides that contain a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides that contain a 4'-2' bridge. Examples of such 4'-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,281); 83); 4'-CH2-N(OCH3)-2' (and analogs; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-O-N(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004/0171570); 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated by reference herein.

Additional representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. No. 6,268,490; U.S. Pat. No. 6,525,191; U.S. Pat. No. 6,670,461; U.S. Pat. No. 6,770,748; U.S. Pat. No. 6,794,499; U.S. Pat. No. 6,998,484; U.S. Pat. No. 7,053,207; U.S. Pat. No. 7,034,133; U.S. Pat. No. 7,084,125; and U.S. Pat. No. 7,399,845. No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; U.S. Patent Application Publication No. 2008/0039618; and U.S. Patent Application Publication No. 2009/0012281 (the entire contents of each of which are hereby incorporated by reference herein).

Any of the aforementioned bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of the iRNA may also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as "S-cEt."

The iRNA of the invention may also include one or more "conformationally locked nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. The CRNs lock the ribose ring into a stable configuration, increasing hybridization affinity to the mRNA. The linker is long enough to place the oxygen in an optimal position for stability and affinity, resulting in less puckering of the ribose ring.

Representative references that teach the preparation of some of the above-mentioned CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013/0190383; and WO 2013/036868, the entire contents of each of which are hereby incorporated by reference herein.

One or more of the nucleotides of the iRNA of the present invention may also include a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as an "unlocked nucleic acid" ("UNA") modification.

Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. No. 8,314,227; and U.S. Patent Application Publication No. 2013/0096289; U.S. Pat. No. 2013/0011922; and U.S. Pat. No. 2011/0313020, the entire contents of each of which are hereby incorporated by reference herein.

Potential stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, and inverted base dT (idT). Disclosure of this modification is found in WO 2011/005861.

A. Modified iRNAs Containing the Motifs of the Invention
In certain aspects of the present invention, the double-stranded RNAi agent of the present invention comprises an agent having chemical modifications, for example, as disclosed in International Publication WO 2013/075035, filed November 16, 2012, the entire contents of which are incorporated herein by reference.As shown herein and in International Publication WO 2013/075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense and/or antisense strands of the RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense and antisense strands of the RNAi agent may be completely modified otherwise.The introduction of these motifs, if present, disrupts the modification pattern of the sense and/or antisense strands.The RNAi agent may optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand.The resulting RNAi agent exhibits excellent gene silencing activity.

More specifically, it has been unexpectedly found that fully modifying the sense and antisense strands of a double-stranded RNAi agent to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent significantly enhances the gene silencing activity of the RNAi agent.

Thus, the present invention provides a double-stranded RNAi agent capable of inhibiting expression of a target gene (i.e., the apolipoprotein C3 (APOC3) gene) in vivo. The RNAi agent includes a sense strand and an antisense strand. Each strand of the RNAi agent may be 12-30 nucleotides in length. For example, each strand may be 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense and antisense strands typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of the RNAi agent may be 12-30 nucleotide pairs long. For example, the duplex region may be 14-30 nucleotide pairs long, 17-30 nucleotide pairs long, 27-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-21 nucleotide pairs long, 17-19 nucleotide pairs long, 19-25 nucleotide pairs long, 19-23 nucleotide pairs long, 19-21 nucleotide pairs long, 21-25 nucleotide pairs long, or 21-23 nucleotide pairs long. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides long.

In one embodiment, the RNAi agent may include one or more overhang regions and/or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. The overhangs may be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhangs may form a mismatch with the target mRNA, or the overhangs may be complementary to the targeted gene sequence, or may be another sequence. The first and second strands may also be joined by additional bases, e.g., to form a hairpin, or by other non-basic linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be modified or unmodified nucleotides, including but not limited to 2'-sugar modifications, such as 2-F, 2'-O methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be an overhang sequence on either end of either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or can be another sequence.

The 5'- or 3'-overhang of the sense strand, the antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, one or more overhang regions include two nucleotides with a phosphorothioate between them, where the two nucleotides may be the same or different. In one embodiment, the overhang is present at the 3' end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which may enhance the interference activity of RNAi without affecting its overall stability. For example, this single-stranded overhang may be located at the 3'-end of the sense strand or at the 3'-end of the antisense strand. The RNAi may also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand), or vice versa. In general, the antisense strand of the RNAi has a nucleotide overhang at the 3'-end and a blunt 5'-end. Without wishing to be bound by theory, an asymmetric blunt end at the 5'-end of the antisense strand and a 3'-end overhang at the antisense strand favors the guide strand loading into the RISC process.

In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

In another embodiment, the RNAi agent is a double-ended bluntmer 20 nucleotides in length, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

In yet another embodiment, the RNAi agent is a double-ended bluntmer 21 nucleotides in length, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, where one end of the RNAi agent is blunt ended and the other end contains a two nucleotide overhang. Preferably, the two nucleotide overhang is at the 3' end of the antisense strand.

When a two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, every nucleotide of the sense and antisense strands of the RNAi agent is a modified nucleotide, including nucleotides that are part of a motif. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand, preferably GalNAc3.

In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues long and, beginning at the 5'-terminal nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1-23 of the first strand; the antisense strand is 36-66 nucleotide residues long and, beginning at the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1-23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; the 5' end of the antisense strand comprises 10-30 consecutive nucleotides which are unpaired with the sense strand. When the sense and antisense strands are aligned for maximum complementarity, at least the 5'- and 3'-terminal nucleotides of the sense strand base pair with nucleotides of the antisense strand, thereby forming a substantially duplexed region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of these motifs is at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand having a length of 25 to 29 nucleotides, and a second strand having a length of 30 nucleotides or less, with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1-4 nucleotides longer than the first strand at its 3' end, the duplex region is at least 25 nucleotides long, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length, such that when the RNAi agent is introduced into a mammalian cell, it reduces target gene expression, and Dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of which is present at the cleavage site of the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of these motifs is at or near the cleavage site of the antisense strand.

For RNAi agents with a duplex region 17-23 nucleotides long, the cleavage sites of the antisense strand are typically at about positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand (counting begins from the first nucleotide from the 5' end of the antisense strand, or counting begins from the first paired nucleotide from the 5' end within the duplex region of the antisense strand). The cleavage site of the antisense strand can also vary depending on the length of the duplex region of the RNAi from the 5' end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the site of the strand break; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of the strand break. When the sense and antisense strands form a dsRNA duplex, the sense and antisense strands may be aligned such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand base pairs with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may be at or near the site of cleavage of the strand, and the other motif may be a wing modification. The term "wing modification" as used herein refers to a motif that is present in another portion of the strand that is separated from a motif that is at or near the site of cleavage of the same strand. The wing modification is either adjacent to the first motif or separated by at least one nucleotide or more. When the motifs are immediately adjacent to each other, the chemistry of the motifs is different from each other, and when the motifs are separated by one or more nucleotides, their chemistry may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may be present on one side of the first motif that is at or near the site of cleavage, or may be present on both sides of the lead motif.

Like the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, at least one of which may be at or near the site of strand cleavage. The antisense strand may also contain one or more wing modifications aligned similarly to the wing modifications that may be present on the sense strand.

In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can be located at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands may be aligned such that two modifications from one strand each are located at one end of the duplex region with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand each are located at the other end of the duplex region with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand are located on either side of the lead motif with an overlap of 1, 2, or 3 nucleotides in the duplex region.

In one embodiment, every nucleotide of the sense and antisense strands of an RNAi agent may be modified, including nucleotides that are part of a motif. Each nucleotide may be modified by the same or different modifications, which may include one or more modifications of one or both of the non-linked and/or linked phosphate oxygens; modifications of components of the ribose sugar, such as the 2' hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with a "dephospho" linker; modifications or replacement of naturally occurring bases; and replacement or modification of the ribose phosphate backbone.

Because nucleic acids are polymers of subunits, many of the modifications are present at positions that are repeated within the nucleic acid, e.g., modifications of the base, or the phosphate moiety, or the non-linked O of the phosphate moiety. In some cases, the modifications may be present at all of the positions of interest within the nucleic acid, but in many cases this is not the case. As an example, the modifications may only be present at the 3' or 5' terminal positions, or may only be present in the terminal regions, e.g., positions on the terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of the strand. The modifications may be present in the double-stranded regions, the single-stranded regions, or both. The modifications may only be present in the double-stranded regions of the RNA, or may only be present in the single-stranded regions of the RNA. For example, phosphorothioate modifications at non-linked O positions may only be present at one or both ends, or may only be present in the terminal regions, e.g., positions on the terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may only be present in the double-stranded and single-stranded regions, especially at the ends. One or more 5' ends may be phosphorylated.

For example, to enhance stability, it may be possible to include certain bases in the overhang or to include modified nucleotides or nucleotide surrogates in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of these bases in the 3' or 5' overhang may be modified, e.g., by the modifications described herein. Modifications may include modifications known in the art, e.g., modifications at the 2' position of the ribose sugar by using modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, in place of the ribosugar of the nucleobase, and modifications at the phosphate group, e.g., the use of phosphorothioate modifications. The overhang need not be homologous to the target sequence.

In one embodiment, each residue in the sense and antisense strands is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The strands may contain more than one modification. In one embodiment, each residue in the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro.

There are typically at least two different modifications on the sense and antisense strands. The two modifications may be 2'-O-methyl or 2'-fluoro modifications, etc.

In one embodiment, Na and/or Nb include an alternating pattern of modifications. The term "alternating motif" as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. Alternating nucleotides may refer to every other nucleotide or every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAAABAAAB...", "AAABBBBAAABBB...", or "ABCABCABCABC...", etc.

The types of modifications included in the alternating motif may be the same or different. For example, if each of A, B, C, D represents one type of modification on a nucleotide, the alternation pattern, i.e. the modifications on every other nucleotide, may be the same, but each of the sense or antisense strands may be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCCD...".

In one embodiment, the RNAi agents of the invention include one in which the modification pattern of the alternating motif on the sense strand is shifted relative to the modification pattern of the alternating motif on the antisense strand. The shift can be such that modified groups of nucleotides in the sense strand correspond to differently modified groups of nucleotides in the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif of the sense strand may begin with "ABABAB" at the 5'-3' end of the strand and the alternating motif of the antisense strand may begin with "BABABA" at the 5'-3' end of the strand within the duplex region. As another example, within a duplex region, the alternating motif in the sense strand may begin with "AABBAABB" at 5'-3' of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" at 5'-3' of the strand, thus resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the sense strand that initially has a shift relative to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the antisense strand, i.e., 2'-O-methyl modified nucleotides on the sense strand base pair with 2'-F modified nucleotides on the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

The introduction of one or more motifs of three identical modifications over three consecutive nucleotides to the sense and/or antisense strand disrupts the original modification pattern present in the sense and/or antisense strand. This disruption of the modification pattern of the sense and/or antisense strand by the introduction of one or more motifs of three identical modifications over three consecutive nucleotides to the sense and/or antisense strand unexpectedly enhances gene silencing activity against the target gene.

In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced to either of these strands, the modification of the nucleotides adjacent to the motif is a different modification than the motif's modification. For example, the portion of the sequence containing the motif is "...NaYYYNb..." [where "Y" represents the modification of the motif of three identical modifications on three consecutive nucleotides, and "Na" and "Nb" represent modifications to the nucleotides adjacent to the motif "YYY" that are different from the modification of Y, and Na and Nb may be the same or different modifications]. Alternatively, Na and/or Nb may be present or absent when a wing modification is present.

The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present on any nucleotide of the sense strand or the antisense strand or both strands at any position of the strand. For example, the internucleotide linkage modification may be present at every nucleotide on the sense strand and/or the antisense strand; each internucleotide linkage modification may be present in an alternating pattern on the sense strand and/or the antisense strand; or the sense strand or the antisense strand may both contain internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region may contain two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between them. Internucleotide bond modifications may also be made to link the overhang nucleotide with the terminal paired nucleotide in the duplex region. For example, at least two, three, four, or all of the overhang nucleotides may be linked via phosphorothioate or methylphosphonate internucleotide bonds, and optionally there may be an additional phosphorothioate or methylphosphonate internucleotide bond linking the overhang nucleotide with the paired nucleotide adjacent to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, two of these three nucleotides being overhang nucleotides and the third nucleotide being the paired nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides may be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and/or the 5' end of the antisense strand.

In one embodiment, the two-nucleotide overhang is at the 3' end of the antisense strand and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of these three nucleotides are overhanging nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent may further have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

In one embodiment, the RNAi agent contains mismatches with the target in the duplex, or combinations thereof. The mismatches can be in the overhang regions or in the duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of association or dissociation of a particular pairing, the simplest approach is to look at each pair for each individual base pair, but next-neighbor or similar analyses can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or non-canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over canonical pairings.

In one embodiment, the RNAi agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs from the 5' end within the duplex region of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

In one embodiment, the nucleotide at position 1 from the 5' end in the duplex region of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs from the 5' end in the duplex region of the antisense strand is an AU base pair. For example, the first base pair from the 5' end in the duplex region of the antisense strand is an AU base pair.

In another embodiment, the 3'-terminal nucleotide of the sense strand is deoxythymine (dT). In another embodiment, the 3'-terminal nucleotide of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, at the 3'-end of the sense strand and/or antisense strand.

In one embodiment, the sense strand sequence has formula (I):
5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'(I)
[Wherein:
i and j are each independently 0 or 1;
p and q are each independently 0 to 6;
each Na independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides;
each Nb independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides;
each np and nq independently represents an overhanging nucleotide;
wherein Nb and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably, YYY are all 2'-F modified nucleotides.

In one embodiment, Na and/or Nb include an alternating pattern of modifications.

In one embodiment, the YYY motif is present at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region 17-23 nucleotides in length, the YYY motif can be present at or near the cleavage site of the sense strand (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13) (counting starting from the first nucleotide from the 5' end; or optionally, counting starting from the first paired nucleotide from the 5' end within the duplex region).

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the formula:
5'np-Na-YYY-Nb-ZZZ-Na-nq3'(Ib);
5'np-Na-XXX-Nb-YYY-Na-nq3'(Ic); or 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3' (Id)
It can be expressed as:

When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), Nb represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each Nb independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6. Each Na may independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand has the formula:
5'np-Na-YYY-Na-nq3'(Ia)
It can be represented by:

When the sense strand is represented by formula (Ia), each Na can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi has the formula (II):
5'nq'-Na'-(Z'Z'Z')k-Nb'-Y'Y'Y'-Nb'-(X'X'X')l-Na'-np'3'(II)
[Wherein:
k and l are each independently 0 or 1;
p' and q' are each independently 0 to 6;
each Na' independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides;
each Nb' independently represents an oligonucleotide sequence containing from 0 to 10 modified nucleotides;
each np' and nq' independently represents an overhanging nucleotide;
where Nb' and Y' do not have the same modification; and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, Na' and/or Nb' include an alternating pattern of modifications.

The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region 17-23 nucleotides in length, the Y'Y'Y' motif can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand (counting starting from the first nucleotide from the 5' end; or optionally, counting starting from the first paired nucleotide from the 5' end within the duplex region). Preferably, the Y'Y'Y' motif is present at positions 11, 12, 13.

In one embodiment, all of the Y'Y'Y' motifs are 2'-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

Thus, the antisense strand has the following formula:
5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Na'-np'3'(IIb);
5'nq'-Na'-Y'Y'Y'-Nb'-X'X'X'-np'3'(IIc); or 5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Nb'-X'X'X'-Na'-np'3' (IId)
It can be represented by:

When the antisense strand is represented by formula (IIb), Nb' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), Nb' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, k is 0 and l is 0, and the antisense strand has the formula:
5'np'-Na'-Y'Y'Y'-Na'-nq'3' (Ia)
It can be represented by:

When the antisense strand is represented as formula (IIa), each Na' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

Each of X', Y' and Z' may be the same as or different from each other.

Each nucleotide of the sense and antisense strands may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain a YYY motif present at positions 9, 10, and 11 of the strand when the duplex region is 21 nt (counting starting from the first nucleotide from the 5' end, or optionally counting starting from the first paired nucleotide from the 5' end within the duplex region); and Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

In one embodiment, the antisense strand may contain a Y'Y'Y' motif present at positions 11, 12, 13 of the strand (counting starting from the first nucleotide from the 5' end, or optionally counting starting from the first paired nucleotide from the 5' end within the duplex region); and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId).

Thus, the RNAi agents used in the methods of the invention can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex has the formula (III):
Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'
Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5'
(III)
[Wherein:
i, j, k, and l are each independently 0 or 1;
p, p', q, and q' are each independently 0 to 6;
each Na and Na' independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two differently modified nucleotides;
each Nb and Nb' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides;
wherein each np', np, nq', and nq (each of which may be present or absent) independently represents an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.
It is represented by:

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or i and j are both 0; or i and j are both 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or k and l are both 0; or k and l are both 1.

Exemplary combinations of sense and antisense strands that form an RNAi duplex include the following formulas:
5'np-Na-YYY-Na-nq3'
3'np'-Na'-Y'Y'Y'-Na'nq'5'
(IIIa)
5'np-Na-YYY-Nb-ZZZ-Na-nq3'
3'np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5'
(IIIb)
5'np-Na-XXX-Nb-YYY-Na-nq3'
3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq'5'
(IIIc)
5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3'
3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq'5'
(IIId)

When the RNAi agent is represented by formula (IIIa), each Na independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When an RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When an RNAi agent is represented as formula (IIIc), each Nb, Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When an RNAi agent is represented as formula (IIId), each Nb, Nb' independently represents an oligonucleotide sequence that includes 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence that includes 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na', Nb, Nb' independently includes an alternating pattern of modifications.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same as or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides are base-paired with a corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired with a corresponding Y' nucleotide.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with a corresponding Z' nucleotide; or all three of the Z nucleotides are base-paired with a corresponding Z' nucleotide.

When an RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides. Alternatively, at least two of the X nucleotides are base-paired with a corresponding X' nucleotide; or all three of the X nucleotides are base-paired with a corresponding X' nucleotide.

In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and/or the modification on the X nucleotide is different from the modification on the X' nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated with one or more GalNAc derivatives (described below) attached via a bivalent or trivalent branched linker. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand includes at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0 and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand includes at least one phosphorothioate bond, and the sense strand is conjugated with one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes may target the same gene or two different genes; or each of the duplexes may target the same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the duplexes being linked by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes may target the same gene or two different genes; or each of the duplexes may target the same gene at two different target sites.

In one embodiment, two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to one another at one or both of the 5' and 3' ends, and are optionally conjugated to a ligand. Each of the agents may target the same gene or two different genes; or each of the agents may target the same gene at two different target sites.

In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modifications, e.g., 10 or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides containing 2'-fluoro modifications. In a specific embodiment, the RNAi agent of the present invention contains 10 nucleotides containing 2'-fluoro modifications, e.g., 4 nucleotides containing 2'-fluoro modifications in the sense strand and 6 nucleotides containing 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides containing 2'-fluoro modifications, e.g., 4 nucleotides containing 2'-fluoro modifications in the sense strand and 2 nucleotides containing 2'-fluoro modifications in the antisense strand.

In other embodiments, the RNAi agents of the present invention may contain very few nucleotides containing 2'-fluoro modifications, e.g., 2 or fewer nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 2, 1, or 0 nucleotides containing 2'-fluoro modifications. In a specific embodiment, the RNAi agent may contain 2 nucleotides containing 2'-fluoro modifications, e.g., 0 nucleotides containing 2-fluoro modifications in the sense strand and 2 nucleotides containing 2'-fluoro modifications in the antisense strand.

Various publications describe multimeric RNAi agents that may be used in the methods of the invention. Such publications include WO 2007/091269, U.S. Pat. No. 7,858,769, WO 2010/141511, WO 2007/117686, WO 2009/014887, and WO 2011/031520, the entire contents of each of which are hereby incorporated by reference.

As described in more detail below, an RNAi agent containing one or more carbohydrate moieties conjugated to the RNAi agent may optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety may be added to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent may be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replaced modified subunit (RRMS). The cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system or may contain two or more rings, e.g., a fused ring. The cyclic carrier may be a fully saturated ring system or may contain one or more double bonds.

Ligands may be attached to polynucleotides via carriers. The carriers include (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tethering attachment point". "Backbone attachment point", as used herein, refers to a functional group, e.g., a hydroxyl group, or generally, a bond available and suitable for incorporation of the carrier into a backbone, e.g., the phosphate backbone of a ribonucleic acid, or a modified phosphate backbone, e.g., a sulfur-containing backbone. "Tethering attachment point" (TAP) refers in some embodiments to a ring atom, e.g., a carbon atom or a heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the moiety of choice is attached. This moiety may be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the moiety of choice is attached to the cyclic carrier by an intervening tethering. Thus, the cyclic carriers often include a functional group, e.g., an amino group, or generally, will provide a bond suitable for incorporation or tethering of another chemical entity, e.g., a ligand, to the ring.

The RNAi agent may be conjugated to the ligand via a carrier, where the carrier may be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

In certain specific embodiments, the RNAi agent used in the methods of the invention is an agent selected from the group of agents listed in any one of Tables 4A, 4B, 5, 8, 9, 10, 11A, 11B, 12, and 13. These agents may further comprise a ligand.

IV. Ligand-conjugated iRNA
Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060); for example, beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., al., Nucl. Acids Res., 1992, 20:533-538); aliphatic chains such as, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moieties (Mishra et al., al., Biochim. Biophys. Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, the ligand alters the distribution, targeting or lifetime of the iRNA agent in which it is incorporated. In a preferred embodiment, the ligand provides improved affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a subcellular or organ compartment, a tissue or organ or region of the body, e.g., compared to a species in which such a ligand is absent. Preferred ligands do not participate in double-stranded pairing in duplexed nucleic acids.

Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, e.g., synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polyamino acids such as polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines are polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or alpha helical peptides.

The ligand may also include a targeting group such as a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody that binds to a specific cell type, e.g., a kidney cell. The targeting group may be thyroid stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetylglucoseamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithochol. acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ conjugates of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

The ligand can be a protein, e.g., a glycoprotein; or a peptide, e.g., a molecule with specific affinity for a co-ligand; or an antibody, e.g., an antibody that binds to a specified cell type, e.g., a liver cell. Ligands may also include hormones and hormone receptors. They may also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Ligands may be, e.g., lipopolysaccharide, p38 MAP kinase activator, or NF-κB activator.

The ligand can be a substance, such as a drug, that can increase uptake of the iRNA agent into the cell, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments, e.g., by disrupting the cell's cytoskeleton. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, the ligand attached to the iRNA described herein refers to a pharmacokinetic regulator (PK regulator). PK regulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK regulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins, and thus short oligonucleotides, such as, for example, about 5-, 10-, 15-, or 20-base oligonucleotides containing multiple phosphorothioate linkages in the backbone, are also suitable for use as ligands (e.g., as PK-modulating ligands) in the present invention. In addition, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of oligonucleotides bearing pendant reactive functional groups, such as those derived from the addition of a binding molecule onto the oligonucleotide (described below). The reactive oligonucleotides may be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing an attached binding moiety.

The oligonucleotides used in the conjugates of the invention may be conveniently and routinely produced through well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including Applied Biosystems (Foster City, Calif.). Additionally or alternatively, any other means for such synthesis known in the art may be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing a ligand molecule, or building blocks bearing non-nucleoside ligands.

When using a nucleotide conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is typically completed and then a ligand molecule is reacted with the linking moiety to produce the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule.Such lipid or lipid-based molecule is preferably bound to serum protein, such as human serum albumin (HSA).HSA-binding ligand allows the distribution of the conjugate to target tissue, such as non-renal target tissue of the body.For example, the target tissue can be the liver, including the liver parenchymal cells.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the degradation resistance of the conjugate, (b) increase the targeting or transport to the target cell or cell membrane, and/or (c) be used to regulate the binding of serum protein, such as HSA.

A lipid-based ligand may be used for inhibition, e.g., to control binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds more weakly to HSA may be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity such that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed.

In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate is preferably distributed to the kidney. Other moieties that target renal cells may also be used in place of or in addition to the lipid-based ligand.

In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, e.g., folate, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by a target cell, e.g., liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Penetration Agents In another aspect, the ligand is a cell penetration agent, preferably a helical cell penetration agent. Preferably, the cell penetration agent is amphipathic. Exemplary cell penetration agents are peptides, such as tat or antennopedia. If the cell penetration agent is a peptide, it may be subject to modifications, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an α-helical agent having a lipophilic and lipophobic phase.

The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. The addition of peptides and peptidomimetics to an iRNA agent can affect the pharmacokinetic distribution of the iRNA, such as by facilitating cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, such as, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

The peptide or peptidomimetic may be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a constrained peptide, or a crosslinked peptide. In another alternative, the peptide moiety may contain a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 24). RFGF analogs containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 25)) may also be targeting moieties. The peptide moiety may be a "delivery" peptide that may transport a number of polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:26)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:27)) have been shown to be capable of functioning as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage-display libraries, or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic that is tethered to a dsRNA agent through an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. The peptide portion can range from about 5 amino acids to about 40 amino acids in length. The peptide portion may have structural modifications to increase stability or induce conformational properties. Any of the structural modifications described below may be used.

RGD peptides used in the compositions and methods of the invention may be linear or cyclic and may be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissues. RGD-containing peptides and peptidiomimetics include D-amino acids, as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands may be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A "cell penetrating peptide" is capable of penetrating a cell, e.g., a microbial cell, e.g., a bacterial or fungal cell, or a mammalian cell, e.g., a human cell. A microbial cell penetrating peptide can be, e.g., an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two key amino acids (e.g., PR-39 or indolicidin). A cell penetrating peptide can also contain a nuclear localization signal (NLS). For example, a cell penetrating peptide can be a bisected amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Complex In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound that has as part of it a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include saccharides AGT and above (e.g., AGT, C6, C7, or C8); di- and trisaccharides include saccharides having two or three monosaccharide units (e.g., AGT, C6, C7, or C8).

In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is
and the like.

In another embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention comprises
is selected from the group consisting of:

Further exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to,
(Formula XXIII) [When one of X or Y is an oligonucleotide, the other is hydrogen].

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, including, but not limited to, a PK modulator and/or a cell penetrating peptide.

Further carbohydrate conjugates suitable for use in the present invention include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers In some embodiments, the conjugates or ligands described herein may be attached to the iRNA oligonucleotide by a variety of linkers, which may be cleavable or non-cleavable.

The term "linker" or "linking group" means an organic moiety that connects two portions of a compound, e.g., by covalently bonding the two portions of a compound. A linker is typically a direct bond, or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroaryl Alkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl , alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, etc., in which one or more methylenes may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-16 atoms, or 8-16 atoms.

A cleavable tether is one that is sufficiently stable outside a cell, but is cleaved upon entry into a target cell to release the two moieties that the linker tethers. In preferred embodiments, the cleavable tether is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more times, or at least about 100 times more rapidly in a target cell or under a first standard condition (e.g., which may be selected to mimic or correspond to intracellular conditions) than in the subject's blood or under a second standard condition (e.g., which may be selected to mimic or correspond to conditions found in blood or serum).

Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity in cells than in serum or blood. Examples of such degradable agents include redox agents selected for a specific substrate or with no substrate specificity, including oxidizing or reducing enzymes or reducing agents such as mercaptans, present in cells that can degrade redox-cleavable linking groups, for example, by reduction; esterases; agents that can result in an acidic environment, such as endosomes or those that result in a pH of 5 or less; enzymes that can act as general acids, peptidases (which can be substrate specific), and phosphatases, thereby hydrolyzing or degrading acid-cleavable linking groups.

Cleavable linking groups such as disulfide bonds can be highly sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand in the cell or to a desired compartment of the cell.

The linker may include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell being targeted. For example, a liver-targeting ligand may be linked to a cationic lipid through a linker that includes an ester group. Hepatocytes are rich in esterases, and therefore the linker is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

Linkers containing peptide bonds can be used to target peptidase-rich cell types such as hepatocytes and synovial cells.

In general, the suitability of candidate cleavable linkers may be evaluated by testing the ability of degradable agents (conditions) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility of cleavage between first and second conditions may be determined, where the first condition is selected to indicate cleavage in target cells and the second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation may be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform an initial evaluation in a cell-free or culture condition and confirm with further evaluation in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox-Cleavable Linkers In one embodiment, the cleavable linker is a redox-cleavable linker that is cleaved upon reduction or oxidation. One example of a reductive cleavable linker is a disulfide linker (-S-S-). To determine whether a candidate cleavable linker is a suitable "reductive cleavable linker" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can resort to the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT), or other reducing agents, using reagents known in the art that mimic the cleavage rates observed in cells, such as target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. One candidate compound is cleaved at most about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound may be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium compared to conditions selected to mimic the extracellular medium.

ii. Phosphate-based cleavable linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker. A phosphate-based cleavable linker can be cleaved by an agent that degrades or hydrolyzes the phosphate group. One example of an agent that cleaves a phosphate group in a cell is an enzyme, such as an intracellular phosphatase. Examples of phosphate based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates may be evaluated using methods similar to those described above.

iii. Acid-cleavable linkers In another embodiment, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less) or by an agent such as an enzyme that can act as a general acid. Within a cell, certain low pH organelles such as endosomes and lysosomes may provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon is attached to the oxygen of the ester (alkoxy group), it is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates may be evaluated using methods similar to those described above.

iv. Ester-Based Linkers In another embodiment, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved intracellularly by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates may be evaluated using methods similar to those described above.

v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved by enzymes, such as peptidases and proteases, within cells. Peptide-based cleavable linkers are peptide bonds that form between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond that forms between amino acids to give rise to peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) that form between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable tethers have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

In one embodiment, the iRNA of the invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates containing linkers of the compositions and methods of the invention include, but are not limited to,
[When one of X and Y is an oligonucleotide, the other is hydrogen].

In certain embodiments of the compositions and methods of the invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

In one embodiment, the dsRNA of the invention has the formula (XXXII)-(XXXV):
[Wherein:
q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represent 0 to 20 for each occurrence, and the repeat units may be the same or different;
P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently, for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O;
Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C, independently at each occurrence, are absent, alkylene, substituted alkylene, where one or more methylenes may be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R')=C(R"), C≡C, or C(O);
R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, and R5C each independently represent, for each occurrence, none, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=N-O,
or heterocyclyl;
L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent a ligand; i.e., each independently at each occurrence represents a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and Ra is H or an amino acid side chain. The trivalent conjugated GalNAc derivative is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any one of the following:
wherein L5A, L5B and L5C represent monosaccharides such as GalNAc derivatives are particularly useful for use in RNAi agents to inhibit expression of target genes.

Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures listed above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA complexes include U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; and 5,218,105, each of which is hereby incorporated by reference in its entirety. US Patent Nos. 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5 ,082,830; U.S. Pat. No. 5,112,963; U.S. Pat. No. 5,214,136; U.S. Pat. No. 5,245,022; U.S. Pat. No. 5,254,469; U.S. Pat. No. 5,258,506; U.S. Pat. No. 5,262,536; U.S. Pat. No. 5,272,250; U.S. Pat. No. 5,292,873; U.S. Pat. No. 5,317,098; U.S. Pat. Nos. 5,371,241, 5,391,723; U.S. Pat. Nos. 5,416,203, 5,451,463; U.S. Pat. No. 5,510,475; U.S. Pat. No. 5,512,667; U.S. Pat. No. 5,514,785; U.S. Pat. 552; U.S. Pat. No. 5,567,810; U.S. Pat. No. 5,574,142; U.S. Pat. No. 5,585,481; U.S. Pat. No. 5,587,371; U.S. Pat. No. 5,595,726; U.S. Pat. No. 5,597,696; U.S. Pat. No. 5,599,923; U.S. Pat. Nos. 5,599,928 and 5,688,941; U.S. Pat. No. 6,294,664; U.S. Pat. No. 6,320,017; U.S. Pat. No. 6,576,752; U.S. Pat. No. 6,783,931; U.S. Pat. No. 6,900,297; U.S. Pat. No. 7,037,646, U.S. Pat. No. 8,106,022, but are not limited thereto.

Not all positions in a given compound need be uniformly modified; in fact, more than one of the aforementioned modifications may be incorporated in a single compound, or even in a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

"Chimeric" iRNA compounds or "chimeras", in the context of the present invention, are iRNA compounds, preferably dsRNA, that contain two or more chemically distinct regions, each composed of at least one monomer unit, i.e., in the case of dsRNA compounds, nucleotides. These iRNAs typically contain at least one region in which the RNA is modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity to the target nucleic acid. Additional regions of the iRNA may serve as enzyme substrates capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of iRNA inhibition of gene expression. As a result, shorter iRNAs often give comparable results when chimeric dsRNAs are used compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

In some cases, the RNA of an iRNA may be modified by a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to enhance their activity, cellular distribution, or intracellular uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties can be lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., J. Am. Chem. 1999, 103:1111), and the like. al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777). Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA complexes are listed above. A typical conjugation protocol involves the synthesis of an RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out in solution phase, while the RNA is still bound to the solid support, or following RNA cleavage. Purification of the RNA conjugate by HPLC typically gives the pure conjugate.

V. Delivery of the iRNA of the invention Delivery of the iRNA of the invention to a cell, such as a cell in a subject, such as a human subject (e.g., a subject in need thereof, such as a subject with an APOC3-related disease), can be accomplished in several different ways. For example, delivery can be performed by contacting a cell with the iRNA of the invention, either in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition comprising the iRNA, such as a dsRNA, to the subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that encode and induce expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with the iRNA of the present invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO 94/02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to consider for delivering an iRNA molecule include, for example, the biological stability of the delivery molecule, prevention of non-specific effects, and accumulation of the delivery molecule in the target tissue. Non-specific effects of iRNA can be minimized by local administration, such as, for example, direct injection or implantation into tissue or local administration of the formulation. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of systemic tissues that may otherwise be harmed by the agent or degrade the agent, and allows for administration of a lower total dose of the iRNA molecule. Several studies have shown successful gene product knockdown when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al (2004) Retina 24:132-138) and by subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) both showed prevention of neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice could reduce tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and extend survival of tumor-bearing mice (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference can be delivered to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y ... al (2005) J. Neurophysiol. 93:594-602), and to the lungs via intranasal administration (Howard, K. A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). To administer iRNA systemically to treat disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent rapid degradation of dsRNA by endogenous endo- and exo-nucleases. Modification of RNA or pharmaceutical carriers can also allow for targeting of iRNA compositions to target tissues, avoiding undesirable non-specific effects. iRNA molecules can be modified by chemical attachment of lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, iRNAs against ApoB conjugated to lipophilic cholesterol moieties were systemically injected into mice, resulting in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to suppress tumor growth and mediate tumor regression in mouse models of prostate cancer. (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015). In alternative embodiments, the iRNA can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. The positively charged cationic delivery systems facilitate binding of the iRNA molecule (which is negatively charged) and also enhance interactions at the negatively charged cell membrane, allowing for efficient uptake of the iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to the iRNA or derivatized to form vesicles or micelles that encapsulate the iRNA (see, e.g., Kim S. H., et al (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of the iRNA upon systemic administration. Methods for making and administering cationic iRNA complexes are well within the capabilities of one of ordinary skill in the art (see, e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, the contents of which are incorporated by reference in their entireties). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN., et al (2003), supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS., et al (2006) Nature 441:111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int. J. Oncol. 26:1087-1091), polyethyleneimines (Bonnet ME., et al (2008) Pharm. Res. Advance online publication August 16; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, DA., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, for systemic administration, the iRNA is complexed with cyclodextrin. Methods for administration of iRNA and cyclodextrin and pharmaceutical compositions are described in U.S. Pat. No. 7,427,605, the entire contents of which are incorporated herein by reference.

A. Vectors Encoding iRNAs of the Invention APOC3 gene-targeting iRNAs can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al. WO 00/22113; Conrad, WO 00/22114; and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or persistent (weeks to months or longer), depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. The transgene can also be constructed to allow it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

Individual iRNA strands or strands can be transcribed from a promoter on an expression vector. If two separate strands are to be expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, the individual strands of the dsRNA can be transcribed by promoters both located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence such that the dsRNA has a stem-loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, and preferably compatible with vertebrate cells, may be used to generate recombinant constructs for iRNA expression as described herein. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting the desired nucleic acid fragment. Delivery of the iRNA expression vector may be by systemic administration, such as intravenous or intramuscular administration, administration to target cells explanted from the patient and then reintroduced into the patient, or any other means that allows for introduction into the desired target cells.

The iRNA expression plasmid may be transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown targeting different regions of the target RNA over a period of a week or more are also contemplated by the present invention. Successful introduction of the vector into the host cell may be monitored using a variety of known methods. For example, transient transfection may be indicated by a reporter, such as a fluorescent marker, such as green fluorescent protein (GFP). Stable transfection into cells in vitro may be ensured using a marker that provides the transfected cells with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems that may be utilized with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not integrate into the genome of the cell. The constructs may include viral sequences for transfection, if desired. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, e.g., EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure expression of the iRNA in the target cell. Other aspects of the vectors and constructs contemplated are described in more detail below.

Vectors useful for delivering iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cell or tissue. Regulatory elements may be selected to provide either constitutive or regulated/inducible expression.

Expression of iRNA can be precisely regulated using inducible control sequences that are sensitive to specific physiological regulators, such as, for example, circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). Those skilled in the art can select appropriate regulatory/promoter sequences based on the intended use of the iRNA transgene.

Viral vectors containing nucleic acid sequences encoding the iRNA may be used. For example, retroviral vectors may be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid to the patient. More details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells, for example to generate stem cells that are more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

Adenoviruses are also contemplated for use in delivering the iRNA of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes, for example, to airway epithelia. Adenoviruses naturally infect airway epithelia and cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al. , Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); WO 94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing the iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

Adeno-associated virus (AAV) vectors can also be used to deliver the iRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, for example, with either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J. Virol. 1999, the entire disclosure of which is incorporated herein by reference. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; WO 94/13788; and WO 93/24641.

Another viral vector suitable for delivering the iRNA of the invention is a vaccinia virus, e.g., an attenuated vaccinia, such as Modified Virus Ankara (MVA) or NYVAC, a pox virus, e.g., an avipox virus, e.g., fowlpox or canarypox.

The tropism of viral vectors can be modified, if desired, by pseudotyping the vector with coat proteins or other surface antigens from other viruses, or by substituting capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by engineering the vector to express different capsid protein serotypes; see, for example, Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

The pharmaceutical preparation of the vector may include the vector in an acceptable diluent, or may comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery vector, e.g., a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical preparation may include one or more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA of the invention. In one embodiment, provided herein is a pharmaceutical composition containing an iRNA as described herein and a pharma- ceutically acceptable carrier. A pharmaceutical composition containing an iRNA is useful for treating a disease or disorder associated with the expression or activity of the APOC3 gene. Such pharmaceutical compositions are formulated based on the delivery method. An example is a composition formulated for systemic administration via parenteral delivery, for example, by subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, for example, by injection into the brain, such as by continuous pump infusion. The pharmaceutical compositions of the invention can be administered in a dosage sufficient to inhibit the expression of the APOC3 gene. In general, a suitable dose of the iRNA of the invention can range from about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally from about 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.

For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

In another embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg g, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.2 5 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 26 to about 45 mg/kg, about 27 to about 45 mg/kg, about 28 to about 45 mg/kg, about 29 to about 45 mg/kg, about 30 to about 45 mg/kg, about 31 to about 45 mg/kg, about 32 to about 45 mg/kg, about 33 to about 45 mg/kg, about 34 to about 45 mg/kg, about 35 to about 45 mg/kg, about 36 to about 45 mg/kg, about 37 to about 45 mg/kg, about 38 to about 45 mg/kg, about 39 to about 45 mg/kg, about 40 to about 45 mg/kg, about 41 to about 45 mg/kg, about 42 to about 45 mg/kg, about 43 to about 45 mg/kg, about 44 to about 45 mg/kg, about 45 to about 45 mg/kg, about 46 to about 45 mg/kg, about 47 to about 45 mg/kg, about 4 5 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg /kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 2 5 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 up to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to The dose is about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of the invention.

For example, the dsRNA may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5 , 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of the invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 6 to about 50 mg/kg, about 7 to about 50 mg/kg, about 8 to about 50 mg/kg, about 9 to about 50 mg/kg, about 10 to about 50 mg/kg, about 11 to about 50 mg/kg, about 12 to about 50 mg/kg, about 13 to about 50 mg/kg, about 14 to about 50 mg/kg, about 15 to about 50 mg/kg, about 16 to about 50 mg/kg, about 17 to about 50 mg/kg, about 18 to about 50 mg/kg, about 19 to about 50 mg/kg, about 20 to about 50 mg/kg, about 21 to about 50 mg/kg, about 22 to about 50 mg/kg, about 23 to about 50 mg/kg, about 24 to about 50 mg/kg, about 25 to about 50 mg/kg, about 26 to about 50 mg/kg, about 27 to about 50 mg/kg, about 28 to about 50 mg/kg, about 29 ... kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 16 to about 45 mg/kg, about 17 to about 45 mg/kg, about 18 to about 45 mg/kg, about 19 to about 45 mg/kg, about 20 to about 45 mg/kg, about 21 to about 45 mg/kg, about 22 to about 45 mg/kg, about 23 to about 45 mg/kg, about 24 to about 45 mg/kg, about 25 to about 45 mg/kg, about 26 to about 45 mg/kg, about 27 to about 45 mg/kg, about 28 to about 45 mg/kg, about 29 to about 45 mg/kg, about 30 to about 45 mg/kg, about 31 to about 45 mg/kg, about 32 to about 45 mg/kg, about 33 to about 45 mg/kg, about 34 to about 45 mg/kg, about 35 to about 45 mg/kg, about 36 to about 45 mg/kg, about 37 to about 45 mg/kg, about 38 to about 45 mg/kg, about 39 to about 45 mg/kg, about 40 to about 45 mg/kg, about 41 to about 45 mg/kg, kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40 mg /kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg g, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg The dsRNA is administered at a dose of about 10 mg/kg to about 30 mg/kg. Values and ranges intermediate to the recited values are also contemplated to be part of the invention.

For example, the subject may receive about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1 ．． 1.1.2 .3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6 ．． 4.6.5 .6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 1 A single therapeutic dose of iRNA may be administered, for example, subcutaneously or intravenously, such as 8, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of the invention.

In some embodiments, subjects are administered a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0. 975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4 .1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6 ．． 3.6.4 5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, Multiple doses of a therapeutic amount of iRNA, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg, are administered, for example, subcutaneously or intravenously. A multiple dose regimen can include daily administration of a therapeutic amount of iRNA, such as for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more.

In other embodiments, the subject is administered a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0. 975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4 .1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6 ．． 3.6.4 5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, Repeated doses of a therapeutic amount of iRNA, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg, are administered, for example, subcutaneously or intravenously. Repeated dose regimes can include regular administration of a therapeutic amount of iRNA, such as every other day, every third day, every fourth day, twice a week, once a week, every other week, or once a month.

In certain embodiments, for example, when the compositions of the invention comprise dsRNA and lipid as described herein, the subject is administered about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg /kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg /kg to about 10mg/kg, about 1mg/kg to about 5mg/kg, about 1mg/kg to about 10mg/kg, about 1.5mg/kg to about 5mg/kg, about 1 ．． 5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg A therapeutic amount of iRNA may be administered, such as about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5 .2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

In certain embodiments of the invention, for example, when a double-stranded RNAi agent includes a modification (e.g., one or more motifs of three identical modifications on three consecutive nucleotides), e.g., one such motif at or near the cleavage site of the agent, six phosphorothioate linkages, and a ligand, such an agent may be administered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09 mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, , about 0.01 mg/kg to about 0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4 mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg, about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg, about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07 mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about 0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4 mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg, about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07 mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about 0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4 mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg, about 0.0 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg, about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07 mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5 mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg, about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05 mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, or about 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate to the above recited values are also contemplated as part of the invention, for example, the RNAi agent may be administered to a subject at a dose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., the RNAi agent in a pharmaceutical composition, may be about 0.01 mg/kg, 0.0125 mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg, 0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.0 375mg/kg, 0.04mg/kg, 0.0425mg/kg, 0.045mg/kg, 0.0475mg/kg, 0.05mg/kg, 0.0525mg/kg, 0.055mg/kg, 0.0575mg/kg, 0.06mg/kg, 0.0625mg/kg, 0.065mg/kg, 0.0675mg/kg, 0.07mg/kg, 0 ．． 0725mg/kg, 0.075mg/kg, 0.0775mg/kg, 0.08mg/kg, 0.0825mg/kg, 0.085mg/kg, 0.0875mg/kg, 0.09mg/kg, 0.0925mg/kg, 0.095mg/kg, 0.0975mg/k g, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg, 0 . It may be administered at a dose of 175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg, 0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45 mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to the aforementioned recited values are also intended to be part of the present invention.

The pharmaceutical composition can be administered by intravenous infusion over a period of time, such as over a period of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about 25 minutes. Administration may be repeated periodically, such as weekly, biweekly (i.e., every two weeks), for one, two, three, four, or more months. After an initial treatment regimen, treatment can be administered less frequently. For example, after weekly or biweekly administration for three months, administration can be repeated monthly for six months or a year or more.

The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three or more partial doses at appropriate intervals throughout the day, or even using continuous infusion or delivery via a controlled release formulation. In that case, the iRNA contained in each partial dose must be correspondingly smaller to achieve the total daily dose. The dosage unit may also be formulated for delivery over several days, for example, using a conventional sustained release formulation that provides sustained release of the iRNA over several days. Sustained release formulations are well known in the art and are particularly useful for site-specific agent delivery, which may be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

In another embodiment, a single dose of the pharmaceutical composition may be administered over an extended period such that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In another embodiment of the invention, a single dose of the pharmaceutical composition of the invention is administered twice a month.

One of skill in the art will appreciate that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and/or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition may include a single treatment or a series of treatments. Effective dosages and in vivo half-lives of individual iRNAs encompassed by the invention may be estimated using conventional procedures or based on in vivo testing using appropriate animal models, as described elsewhere herein.

The pharmaceutical compositions of the invention may be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be topical (e.g., by transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including nebulizers; intratracheal, intranasal, transepidermal and transdermal, oral or parenteral. Parenteral administration may include intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal administration, e.g., via an implanted device; or intracranial administration, e.g., intraparenchymal, intrathecal or intraventricular.

The iRNA can be delivered in a manner that targets a specific tissue, such as the liver (e.g., liver parenchymal cells).

Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is in admixture with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), cationic (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA featured in the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNA can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C1-20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharma- ceutically acceptable salts thereof). Topical formulations are described in detail in U.S. Pat. No. 6,747,014, incorporated herein by reference.

A. iRNA Formulations Comprising Membrane-Like Molecular Assemblies The iRNA used in the compositions and methods of the present invention may be formulated for delivery in membrane-like molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as one bilayer or multiple bilayers. Liposomes include unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the iRNA composition, but may in some cases. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when the liposomes are applied to a tissue, the liposomal bilayer fuses with the bilayer of the cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the iRNA, are delivered into the cell, where the iRNA can specifically bind to and mediate the iRNA targeting. In some cases, the liposomes are also specifically targeted, for example, to direct the iRNA to a particular cell type.

Liposomes containing an iRNA agent may be prepared by a variety of methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed without the lipid components. For example, the lipid components may be amphipathic cationic lipids or lipid complexes. The detergent may have a high critical micelle concentration and may be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the iRNA agent and condense around the iRNA agent to form a liposome. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of the iRNA agent.

If necessary, a carrier compound, e.g., to aid in condensation, can be added during the condensation reaction by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid, e.g., spermine or spermidine. The pH can also be adjusted to aid in condensation.

Methods for generating stable polynucleotide delivery vehicles incorporating polynucleotide/cationic lipid complexes as a structural component of the delivery vehicle are further described, for example, in WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation is described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. The method may also include one or more aspects of the exemplary methods described in Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and a combination of freeze-thawing and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). If consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted for packaging of RNAi agent preparations within liposomes.

Liposomes are divided into two broad classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid/liposome complexes bind to the negatively charged cell surface and are internalized inside endosomes. Due to the acidic pH inside the endosomes, liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

pH-sensitive or negatively charged liposomes do not complex with nucleic acids but rather encapsulate them. Because both nucleic acids and lipids have similar charges, repulsion occurs rather than complexation. Some nucleic acid is still encapsulated in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposome composition contains phospholipids in addition to naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, whereas anionic fusogenic liposomes are formed primarily from dioleoyl sphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC, and egg PC. Another type is formed from a mixture of phospholipids and/or phosphatidylcholine and/or cholesterol.

Other methods for introducing liposomes into cells in vitro and in vivo include those described in U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems, especially those containing non-ionic surfactants and cholesterol, have been studied to determine their utility in drug delivery to the skin. Non-ionic liposomal formulations containing Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A into the dermis of mouse skin. The results suggested that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, which term as used herein refers to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome (A) contains one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the improved circulation half-life of these sterically stabilized liposomes is due to reduced uptake into reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes containing (1) sphingomyelin and (2) ganglioside GM1 or galactocerebroside sulfate. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Non-cationic liposomes cannot fuse efficiently with the plasma membrane, but can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

Additional advantages of liposomes include: Liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; and liposomes can protect iRNA agents encapsulated in their internal compartment from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposomal formulations are the lipid surface charge, vesicle size, and aqueous volume of the liposomes.

The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the plasma membrane of tissue culture cells, resulting in iRNA agent delivery (see, e.g., Felgner, P.L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

The ADOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivery of highly anionic nucleic acids to living tissue culture cells, and it contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complexes thus prepared spontaneously attach to negatively charged cell surfaces and fuse with the plasma membrane, efficiently delivering functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are linked by ester rather than ether bonds.

Other reported cationic lipid compounds include those conjugated to a variety of moieties, including carboxyspermine conjugated to one of two lipid types, such as compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid complex involves derivatization of lipids with cholesterol ("DC-Chol") in combination with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California), and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suitable for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered agent, increased accumulation of the administered agent at the desired target, and the ability to administer the iRNA agent intradermally. In some implementations, liposomes are used to deliver the iRNA agent to epidermal cells and to enhance penetration of the iRNA agent into dermal tissues, e.g., within the skin. For example, liposomes may be applied topically. Topical delivery of therapeutic agents formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al., J. Immunol. 1999, 11:131-135, 1999). al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems, particularly those containing non-ionic surfactants and cholesterol, have been studied to determine their utility in drug delivery to the skin. Non-ionic liposomal formulations containing Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin disorders.

Liposomes containing iRNA can be highly deformable. Such deformability can allow liposomes to penetrate through pores smaller than the average radius of the liposome. For example, transferosomes are one type of deformable liposome. Transfersomes can be created by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing iRNA agents can be delivered subcutaneously, for example, by infection, to deliver the iRNA agent to keratinocytes in the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of a suitable transdermal gradient. Furthermore, due to lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and are often self-loading.

Other formulations to which the present invention may be applied are described in U.S. Provisional Patent Application No. 61/018,616, filed January 2, 2008; U.S. Provisional Patent Application No. 61/018,611, filed January 2, 2008; U.S. Provisional Patent Application No. 61/039,748, filed March 26, 2008; U.S. Provisional Patent Application No. 61/047,087, filed April 22, 2008, and U.S. Provisional Patent Application No. 61/051,528, filed May 8, 2008. PCT application PCT/US2007/080331, filed October 3, 2007, also describes formulations to which the present invention may be applied.

Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate through pores smaller than droplets. Transfersomes can adapt to the environment in which they are used, e.g., they self-optimize (adapt to skin pore shape), self-repair, often reach their target without fragmentation, and are often self-loading. To create transfersomes, a surface edge activator, usually a surfactant, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and ranking the properties of the many different surfactant types, both natural and synthetic, is through the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying the different surfactants used in formulations (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used over a wide pH range. Generally, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most commonly found members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and acyl sulfosuccinates, and acyl phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and soaps.

If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

The use of surfactants in pharmaceuticals, formulations and emulsions has been reviewed (Rieger, Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA used in the methods of the invention may also be provided as a micellar formulation. A "micelle" is defined herein as a particular type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that the hydrophobic portions of the molecules all face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. The reverse arrangement exists if the environment is hydrophobic.

Mixed micelle formulations suitable for delivery through a transdermal membrane may be prepared by mixing an siRNA composition, an alkali metal C8-C22 alkyl sulfate, and an aqueous solution of a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharma- ceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharma- ceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles form when the components are mixed in virtually any way, but to provide smaller micelles, they are mixed vigorously.

In one method, a first micelle composition is prepared containing an siRNA composition and at least an alkali metal alkyl sulfate. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, a micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by the addition of the remaining micelle-forming compounds with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added along with the micelle forming components. An isotonicity agent such as glycerin may also be added after the mixed micelle composition is formed.

To deliver the micellar formulation as a spray, the formulation may be placed in an aerosol dispensing device and the dispensing device may be loaded with the propellant. The propellant, which is under pressure, is in liquid form in the dispensing device. The ratio of the components is adjusted so that the aqueous phase and the propellant phase are one, i.e. one phase. If there are two phases, the dispensing device must be shaken, for example, before dispersing a portion of the contents through the metered valve. The dispensed dose of the pharmaceutical product is ejected from the metered valve in a fine spray.

Propellants include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively simple experimental methods. For absorption through the oral cavity, it is often desirable to increase the dose, e.g., at least two or three times, that for administration through injection or through the gastrointestinal tract.

B. Lipid Particles The iRNA, eg, dsRNA, of the invention can be fully encapsulated in a lipid formulation, eg, LNPs, or other nucleic acid-lipid particles.

As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs typically include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs exhibit long circulatory lifetimes after intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the site of administration), making them extremely useful for systemic applications. LNPs include "pSPLPs" that include encapsulated condensing agent-nucleic acid complexes as described in WO 00/03683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Pat. No. 5,976,567; U.S. Pat. No. 5,981,501; U.S. Pat. No. 6,534,484; U.S. Pat. No. 6,586,410; U.S. Pat. No. 6,815,432; U.S. Patent Application Publication No. 2010/0324120; and WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above cited ranges are also considered part of the invention.

Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl The compound may be 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecane-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane may be used to create lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61/107,998, filed October 23, 2008, which is incorporated herein by reference.

In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (molar percentages), with a particle size of 63.0±20 nm and an siRNA/lipid ratio of 0.027.

Ionic/non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerin (DOPG), dipalmitoylphosphatidylglycerin (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl) The lipid may be an anionic or neutral lipid, including, but not limited to, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

The conjugated lipid that inhibits particle aggregation can be, for example, without limitation, a polyethylene glycol (PEG)-lipid, including PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that inhibits particle aggregation can be from 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, e.g., from about 10 mol% to about 60 mol% or about 48 mol% of the total lipid present in the particle.

In one embodiment, lipidoid ND98·4HCl (MW 1487) (see U.S. Patent Application Serial No. 12/056,230, filed March 26, 2008, the contents of which are incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) may be used to create lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol may be prepared as follows: ND98, 133 mg/ml; cholesterol, 25 mg/ml; PEG-ceramide C16, 100 mg/ml. Stock solutions of ND98, cholesterol, and PEG-ceramide C16 may then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution may be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) to a final ethanol concentration of about 35-45% and a final sodium acetate concentration of about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture may be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using, for example, a thermobarrel extruder such as the Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange may be accomplished, for example, by dialysis or tangential flow filtration. The buffer may be exchanged with phosphate buffered saline (PBS) at about pH 7, for example about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, for example, in WO 2008/042973, which is hereby incorporated by reference.

Further exemplary lipid-dsRNA formulations are listed in Table 1.

Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in WO 2009/127060, filed April 15, 2009, which is incorporated herein by reference.

Formulations containing XTC are described, for example, in U.S. Provisional Application No. 61/148,366, filed January 29, 2009; U.S. Provisional Application No. 61/156,851, filed March 2, 2009; U.S. Provisional Application No. 61/228,373, filed July 24, 2009; U.S. Provisional Application No. 61/239,686, filed September 3, 2009, and International Application No. PCT/US2010/022614, filed January 29, 2010, which are incorporated herein by reference.

Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010/0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference.

Formulations containing ALNY-100 are described, for example, in International Application PCT/US09/63933, filed November 10, 2009, which is incorporated herein by reference.

C12-200-containing formulations are described in U.S. Provisional Patent Application No. 61/175,770, filed May 5, 2009, and International Application No. PCT/US10/33777, filed May 5, 2010, which are incorporated herein by reference.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which the DsRNA featured in the present invention is administered in conjunction with one or more penetration enhancing surfactants and chelating agents. Suitable surfactants include fatty acids and/or esters or their salts, bile acids and/or their salts. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharma- ceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The DsRNA featured in the present invention may be delivered orally in granular form, including spray-dried particles, or complexed to form micro- or nanoparticles. DsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkylcyanoacrylic acids; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylic acids; DEAE-derivatized polyimines, pullulans, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexyl ... acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (intracerebrovascular), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharma- ceutical acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are liver-targeted formulations when treating liver disorders, such as liver cancer.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of combining the active ingredients with pharmaceutical carriers or excipients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Suspensions may also contain stabilizers.

C. Additional Formulations i. Emulsions The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another liquid, typically in the form of droplets greater than 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc. , New York, N. Y. , volume 1, p. 199; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc. , New York, N. Y. , Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc. , New York, N. Y. , volume 2, p. 335; Higuchi et al., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems that contain two immiscible liquid phases that are intimately mixed and dispersed with one another. In general, emulsions can be either water-in-oil (w/o) or oil-in-water (o/w). When the aqueous phase is finely dispersed and dispersed as minute droplets in the bulk oily phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oily phase is finely dispersed and dispersed as minute droplets in the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phase and the active agent, which may be present as a solution in either the aqueous and oily phases or as a separate phase itself. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions that contain more than two phases, such as oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often offer certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion surround small water droplets constitute w/o/w emulsions. Similarly, oil droplet systems that are encapsulated in globules of water and stabilized in an oily continuous phase provide o/w/o emulsions.

Emulsions are characterized by having little or no thermodynamic stability. Frequently, the dispersed or discontinuous phase of an emulsion is well dispersed within the external or continuous phase and is maintained in this form through the means of emulsifiers or formulation viscosity. Either of the emulsion phases may be semisolid or solid, as is the case with emulsion-style ointment bases and creams. Another means of stabilizing emulsions involves the use of emulsifiers, which may be incorporated into either of the emulsion phases. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel (See Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have a wide range of uses in emulsion formulations and are reviewed in the literature (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and contain hydrophilic and hydrophobic moieties. The ratio of hydrophilicity to hydrophobicity is called the surfactant's hydrophilic/lipophilic balance (HLB) and is a useful tool for classifying and selecting surfactants in the preparation of formulations. Surfactants can be divided into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel (See Dekker, Inc., New York, N.Y., volume 1, p. 285).

Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phospholipids, lecithin, and acacia. Absorption bases with hydrophilic properties such as anhydrous lanolin and hydrophilic petrolatum that can take up water to form w/o emulsions yet maintain their semi-solid consistency. Finely dispersed solids are also used as excellent emulsifiers in viscous preparations, especially in combination with surfactants. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids, or hydrocolloids, include natural gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers). These disperse in water or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed phase droplets and by increasing the viscosity of the external phase.

Because emulsions often contain several components such as carbohydrates, proteins, sterols, and phospholipids that can readily support microbial growth, preservatives are often incorporated into these formulations. Commonly used preservatives included in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. The antioxidants used can be free radical scavengers such as tocopherol, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene; or reducing agents such as ascorbic acid and sodium metabisulfite; and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations through dermal, oral, and parenteral routes and methods for their preparation have been reviewed in the literature. (For example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott William ms & Wilkins (8th ed.), New York, NY; Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc. ., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of formulation and efficiency in terms of absorption and bioavailability (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; see Idson, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199. Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutrients are among the materials commonly administered orally as o/w emulsions.

ii. Microemulsions In one embodiment of the present invention, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion may be defined as a system of water, oil, and amphiphiles that is a single optically isotropic and thermodynamically stable solution (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, typically a medium chain length alcohol, to form a transparent system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface active molecules (Leung and Shah, Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions are usually prepared through the combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w/o) or oil-in-water (o/w) depends on the properties of the oil and surfactant used, and the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach using phase diagrams has been extensively studied and provides the skilled artisan with comprehensive knowledge of microemulsion formulation (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; see Block, Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs into a spontaneously formed, thermodynamically stable formulation of droplets.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactants, which are usually short chain alcohols such as ethanol, 1-propanol, and 1-butanol, help increase the interfacial fluidity by penetrating the surfactant coating, thus creating an irregular coating due to the gaps that arise between the surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, aqueous drug solution, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, anticipated enhanced drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides, or iRNA. Microemulsions have been effective in transdermal delivery of active ingredients for both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to facilitate increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract, as well as improve localized cellular uptake of iRNA and nucleic acids.

The microemulsions of the invention may also contain additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve the properties of the formulation and enhance absorption of the iRNA and nucleic acids of the invention. The penetration enhancers used in the microemulsions of the invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

The iRNA agents of the invention may be incorporated into particles, such as, for example, microparticles. Microparticles can be produced by spray drying, but they may also be produced by other methods, including freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration enhancer In one embodiment, the present invention uses various penetration enhancers to provide efficient delivery of nucleic acid, especially iRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, usually only lipid-soluble or lipophilic drugs can easily pass through cell membranes. It has been found that even non-lipophilic drugs can pass through cell membranes if the membrane they pass through is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and nonchelating nonsurfactants (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the aforementioned classes of penetration enhancers is described in more detail below.

Surfactants (or "surface active agents") are chemicals that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in improved absorption of iRNA through mucous membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether) (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol. , 1988, 40, 252).

Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, their C1-20 alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and di-glycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.). (See, e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological roles of bile include facilitating the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38, Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). A variety of naturally occurring bile salts, as well as their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharma- ceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydrofusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE). （例えばＭａｌｍｓｔｅｎ，Ｍ．Ｓｕｒｆａｃｔａｎｔｓ ａｎｄ ｐｏｌｙｍｅｒｓ ｉｎ ｄｒｕｇ ｄｅｌｉｖｅｒｙ，Ｉｎｆｏｒｍａ Ｈｅａｌｔｈ Ｃａｒｅ，Ｎｅｗ Ｙｏｒｋ，ＮＹ，２００２；Ｌｅｅ ｅｔ ａｌ．，Ｃｒｉｔｉｃａｌ Ｒｅｖｉｅｗｓ ｉｎ Ｔｈｅｒａｐｅｕｔｉｃ Ｄｒｕｇ Ｃａｒｒｉｅｒ Ｓｙｓｔｅｍｓ，１９９１，ｐａｇｅ ９２；Ｓｗｉｎｙａｒｄ，Ｃｈａｐｔｅｒ ３９，Ｒｅｍｉｎｇｔｏｎ’ｓ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｓｃｉｅｎｃｅｓ，１８ｔｈ Ｅｄ．，Ｇｅｎｎａｒｏ，ｅｄ．，Ｍａｃｋ Ｐｕｂｌｉｓｈｉｎｇ Ｃｏ．，Ｅａｓｔｏｎ，Ｐａ．，１９９０，ｐａｇｅｓ 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelators as used in the context of the present invention may be defined as compounds that form complexes with metal ions, thereby removing them from solution, resulting in improved iRNA absorption through mucous membranes. With regard to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as deoxyribonuclease inhibitors, since most DNA nucleases require divalent metal ions for catalysis and are inhibited by chelators (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylic acid, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines). （例えばＫａｔｄａｒｅ，Ａ．ｅｔ ａｌ．，Ｅｘｃｉｐｉｅｎｔ ｄｅｖｅｌｏｐｍｅｎｔ ｆｏｒ ｐｈａｒｍａｃｅｕｔｉｃａｌ，ｂｉｏｔｅｃｈｎｏｌｏｇｙ，ａｎｄ ｄｒｕｇ ｄｅｌｉｖｅｒｙ，ＣＲＣ Ｐｒｅｓｓ，Ｄａｎｖｅｒｓ，ＭＡ，２００６；Ｌｅｅ ｅｔ ａｌ．，Ｃｒｉｔｉｃａｌ Ｒｅｖｉｅｗｓ ｉｎ Ｔｈｅｒａｐｅｕｔｉｃ Ｄｒｕｇ Ｃａｒｒｉｅｒ Ｓｙｓｔｅｍｓ，１９９１，ｐａｇｅ ９２；Ｍｕｒａｎｉｓｈｉ，Ｃｒｉｔｉｃａｌ Ｒｅｖｉｅｗｓ ｉｎ Ｔｈｅｒａｐｅｕｔｉｃ Ｄｒｕｇ Ｃａｒｒｉｅｒ Ｓｙｓｔｅｍｓ，１９９０，７，１－３３；Ｂｕｕｒ ｅｔ al., J. Control Rel., 1990, 14, 43-51).

As used herein, a non-chelating, non-surfactant permeation enhancing compound may be defined as a compound that demonstrates insignificant activity as a chelating agent or as a surfactant, but that nevertheless enhances absorption of iRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory agents such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance cellular uptake of iRNA may also be added to the pharmaceutical and other compositions of the invention. For example, cationic lipids such as lipofectin (U.S. Pat. No. 5,705,188 to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (WO 97/30731 to Lollo et al.) are also known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000, among others. CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), iRNAMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Inv itrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; zerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugen e (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx(TM)-20 Reagent(Promega; Madison, WI), Tfx(TM)-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France ), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec(TM)/LipoGen(TM) (Invitrogen; San Diego, CA, USA), P erFectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection Reagent (Genlantis; San D iego, CA, USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; go, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect ( Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B -Bridge International;Mountain Examples include HiFect™ (B-Bridge International, Mountain View, CA, USA) and HiFect™ (B-Bridge International, Mountain View, CA, USA).

Other agents may be utilized to enhance the penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol; pyrroles such as 2-pyrrole; azone; and terpenes such as limonene and menthone.

v. Carrier Certain compositions of the present invention also incorporate a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or an analog thereof, that is inert (i.e., has no biological activity by itself) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example by degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of nucleic acid and carrier compound, typically with an excess of the latter substance, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, possibly due to competition between the carrier compound and the nucleic acid for their normal receptors. For example, recovery of partial phosphorothioate dsRNA in liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic, or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients In contrast to carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharma- ceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid, and are selected with the proposed mode of administration in mind to provide the desired bulk, consistency, etc., when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (such as starch, sodium starch glycolate, etc.); and wetting agents (such as sodium lauryl sulfate, etc.).

The compositions of the present invention may be formulated using pharma- ceutically acceptable organic or inorganic excipients suitable for oral administration that do not adversely react with nucleic acids. Suitable pharma-ceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of the nucleic acid in a liquid or solid oil base. The solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for oral administration that do not adversely react with the nucleic acid may be used.

Suitable pharma- ceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

vii. Other components The compositions of the present invention may further contain other auxiliary components conventionally found in pharmaceutical compositions, at their usage levels established in the art. Thus, for example, the compositions may contain additional compatible pharmacologically active materials, such as antipruritic agents, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful for physically compounding various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, colorants, flavorings and/or aromatic substances, etc.

Aqueous suspensions may contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. Suspensions may also contain stabilizers.

In some embodiments, the pharmaceutical compositions featured herein include (a) one or more iRNA compounds, and (b) one or more agents that function via a non-iRNA mechanism and are useful for treating APOC-related disorders. Examples of such agents include, but are not limited to, anti-inflammatory, anti-adipose, anti-viral, and/or anti-fibrotic agents. In addition, other substances commonly used to protect the liver, such as silymarin, may also be used in combination with the iRNAs described herein. Other agents useful for treating liver disease include telbivudine, entecavir, telaprevir and protease inhibitors such as those disclosed in U.S. Patent Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217 to Tung et al.; and U.S. Patent Application Publication No. 2004/0127488 to Hale et al.

The toxicity and therapeutic efficacy of such compounds may be determined by standard pharmaceutical procedures, e.g., in cell cultures or experimental animals to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

Data obtained from cell culture assays and animal studies may be used to formulate a dosage range for use in humans. Dosages of compositions featured herein are generally within a range of circulating concentrations, including the ED50, that result in little or no toxicity. Dosages may vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods featured herein, a therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range (e.g., achieve a reduction in polypeptide concentration) of the compound, or of the polypeptide product of the target sequence, as appropriate, including the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms), as determined in cell culture. Such information may be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to those administrations discussed above, the iRNAs featured herein may be administered in combination with other known agents effective in treating pathological processes mediated by APOC3 expression. In any event, the treating physician may adjust the amount and timing of iRNA administration based on results observed using standard measures of efficacy known in the art or described herein.

VII. METHODS OF THE PRESENT DISCLOSURE The present invention provides therapeutic and prophylactic methods comprising administering to a subject having or susceptible to developing an APOC3-related disease, disorder, and/or condition (e.g., hypertriglyceridemia) a pharmaceutical composition comprising an iRNA agent of the invention, or a vector containing an iRNA.

In one aspect, the invention provides a method of treating a subject having a disorder that may benefit from reduced APOC3 expression, e.g., hypertriglyceridemia and other APOC-3-related disorders, e.g., nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance); hypertension; cardiovascular disorders, e.g., atherosclerosis; and pancreatitis, e.g., acute pancreatitis.

The therapeutic methods (and uses) of the invention include administering to a subject, e.g., a human, a therapeutically effective amount of an iRNA agent that targets the APOC3 gene or a pharmaceutical composition that includes an iRNA agent that targets the APOC3 gene, thereby treating the subject having a disorder that would benefit from reduced APOC3 expression.

In one aspect, the invention provides a method for preventing at least one symptom in a subject having a disorder that may benefit from reduced APOC3 expression, e.g., an APOC3-related disease, such as hypertriglyceridemia, and other diseases that may be caused by, associated with, or result from hypertriglyceridemia. The latter diseases include, but are not limited to, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovarian syndrome, renal disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis. The method includes administering to the subject a therapeutically effective amount of an iRNA agent, e.g., a dsRNA, or vector, of the invention, thereby preventing at least one symptom in a subject having a disorder that may benefit from reduced APOC3 expression.

In another aspect, the invention provides for the use of a therapeutically effective amount of an iRNA agent of the invention to treat a subject, e.g., a subject that may benefit from reduced and/or inhibited APOC3 expression.

In a further aspect, the invention provides for the use of an iRNA agent, e.g., a dsRNA, or a pharmaceutical composition comprising an iRNA agent, targeting the APOC3 gene, of the invention in the manufacture of a medicament for treating a subject, e.g., a subject who may benefit from reduced and/or inhibited APOC3 expression, e.g., a subject having a disorder that may benefit from reduced APOC3 expression, e.g., an APOC3-related disease, such as hypertriglyceridemia, and other diseases that may be caused by, associated with, or result from hypertriglyceridemia. The latter diseases may include, but are not limited to, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis.

In another aspect, the present invention provides the use of an iRNA, e.g., a dsRNA, of the present invention to prevent at least one symptom in a subject suffering from a disorder that may benefit from reduced and/or inhibited APOC3 expression, e.g., an APOC3-related disease, e.g., hypertriglyceridemia and other diseases that may be caused by, associated with, or result from hypertriglyceridemia. The latter diseases may include, but are not limited to, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis.

In a further aspect, the invention provides the use of an iRNA agent of the invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that may benefit from reduced and/or inhibited APOC3 expression, e.g., an APOC3-related disease, e.g., hypertriglyceridemia and other diseases that may be caused by, associated with, or result from hypertriglyceridemia. The latter diseases may include, but are not limited to, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus (insulin resistance), atherosclerosis, cardiovascular disease, or pancreatitis.

In one embodiment, the iRNA agent targeting APOC3 is administered to a subject having an APOC3-associated disease such that, when the dsRNA agent is administered to the subject, the level of APOC3 in, e.g., cells, tissues, blood or other tissues or bodily fluids of the subject is reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109 5%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more reduction.

The methods and uses of the present invention include administering a composition described herein such that expression of the target APOC3 gene is reduced for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target APOC3 gene is reduced for an extended period of time, e.g., at least about 2, 3, 4, 5, 6, 7 days or more, e.g., about 1 week, 2 weeks, 3 weeks, or about 4 weeks or more.

Administration of dsRNA according to the methods and uses of the invention may result in a reduction in the severity, signs, symptoms, and/or markers of an APOC3-associated disease in a patient having such disease or disorder. In this context, "reduction" refers to a statistically significant decrease in such level. The reduction may be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

The effectiveness of disease treatment or prevention may be assessed, for example, by measuring the level of disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of drug required to sustain the therapeutic effect, disease markers, or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one of ordinary skill in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. For example, the effectiveness of treatment of hypertriglyceridemia may be assessed, for example, by periodic monitoring of blood triglyceride levels. By comparing subsequent readings to the initial reading, the physician has an indication of whether the treatment is effective. It is well within the ability of one of ordinary skill in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. With respect to administration of an iRNA or pharmaceutical composition thereof targeting APOC3, "effective against" an APOC3-related disease indicates that administration in a clinically relevant manner results in a beneficial effect in at least a statistically significant percentage of patients, such as amelioration of symptoms, cure, reduction of disease, extension of life span, improvement in quality of life, or other effect generally recognized as positive by physicians knowledgeable in the treatment of APOC3-related diseases and related causes.

Therapeutic or prophylactic efficacy is evident when there is a statistically significant improvement in one or more parameters of the disease state, or by the lack of a worsening or onset of symptoms expected in the absence of treatment. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more, in a measurable parameter of the disease may indicate effective treatment. The efficacy of a given iRNA agent or combination of agents may also be determined using an experimental animal model for a given disease known in the art. When using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant reduction in a marker or symptom is observed. Suitable animal models of APOC3-related diseases include, for example, any animal model with hypertriglyceridemia. Such animal models include, for example, transgenic mice expressing the human apolipoprotein C2 (APOC2) gene (such as mice of the strain B6;CBA-Tg(APOC2)2Bres/J or B6.Cg-Tg(APOC2)2Bres/J available from Jackson Laboratory, Bar Harbor, Maine); transgenic mice expressing the human apolipoprotein C3 (APOC3) gene (such as mice of the strain B6;CBA-Tg(APOC3)3707Bres/J available from Jackson Laboratory); homozygous fatty liver dystrophy (fld) mice (such as mice of the strain C3H/HeJ-Lpin1fld-2J/J available from Jackson Laboratory, Bar Harbor, Maine); mice homozygous for an ENU-induced missense mutation called Sec61a1Y344H (such as mice of the line C57BL/6J-Sec61a1m1Gek/J, available from Jackson Laboratory); diet-induced obese mice (mice of the line C57BL/6J fed a 60% kcal fat diet, available from Jackson Laboratory, or mice of the line C57BL/6NTac fed a 60% kcal fat diet, available from Taconic); and transgenic C57Bl/6 mice overexpressing the human ApoC3 gene under the liver-specific TBG promoter or as described herein.

In certain embodiments, experimental animal models suitable for testing the efficacy of the iRNA or formulations comprising the iRNA of the present invention include rabbits. Exemplary rabbit models include, for example, Watanabe Hereditary Hyperlipidemic (WHHL) rabbits. WHHL rabbits are an animal model of hypercholesterolemia resulting from a deficiency in the low-density lipoprotein (LDL) receptor. The characteristics of WHHL rabbits and the history of research conducted using WHHL rabbits are described, for example, by Shiomi, M. and Ito, T., Artherosclerosis (2009), 207(1):1-7, the entire contents of which are hereby incorporated by reference herein. WHHL rabbits exhibit increased cholesterol and triglyceride levels, as shown below:

In certain embodiments, WHHL rabbits may be a preferred animal model for studying inhibition of APOC3 expression because they exhibit a more human-like lipid profile compared to other animal models and may contribute to understanding the relationship between ApoC3 knockdown and triglyceride lowering, as well as inform dose determination for studies involving non-human primates. A comparison of enzyme and lipoprotein profiles among various animal species is provided below in Table 2.

Other exemplary rabbit models that may be suitable for testing the efficacy of the iRNA or formulations containing iRNA of the present invention include, for example, diet-induced obese rabbits. For diet-induced obese rabbits, see, for example, Taylor and Fan, Front. Biosci. (1997), 2:298-308; Carroll et al., Am. J. Physiol. (1996), 271:H373-8; Antic et al., Am. J. Hypertens. (2000), 13:556-9; Carroll et al., Acta Physiol. Scand. (2004), 181:183-91; and Long et al., Arterioscler. Thromb. Vasc. Biol. (1999), 19:2179-88, the entire contents of which are incorporated herein by reference.

Subjects were administered approximately 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg g, 0.9mg/kg, 0.95mg/kg, 1.0mg/kg, 1.1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg/kg, 2.0mg/kg, 2 .1mg/kg, 2.2mg/kg, 2.3mg/kg, 2.4mg/kg, 2.5mg/kg dsRNA, 2.6mg/kg dsRNA, 2.7mg/kg dsRNA, 2.8mg /kg dsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1 mg/kg dsRNA, 4 . 2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA NA, 5.6 mg/kg dsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kg dsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kg dsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kg dsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kg dsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg g/kg dsRNA, 8.4 mg/kg dsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kg dsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, Therapeutic amounts of iRNA may be administered, such as 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA, or about 50 mg/kg dsRNA. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

For example, in certain embodiments in which the compositions of the invention comprise a dsRNA as described herein and a lipid, a subject is administered about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0 ．． 2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg /kg to about 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg /kg to about 5mg/kg, about 4mg/kg to about 10mg/kg, about 4.5mg/kg to about 10mg/kg, about 5mg/kg to about 10mg/kg A therapeutic amount of iRNA may be administered, such as about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5 .2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

For example, in another embodiment in which the composition of the invention comprises a dsRNA described herein and N-acetylgalactosamine, a subject is administered about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 ... .5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 4 5 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg g, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 4 ... 0 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3 .5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg A therapeutic amount of iRNA may be administered, such as a dose of about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, when a composition of the invention comprises a dsRNA and N-acetylgalactosamine as described herein, a subject may be administered a therapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

For example, the subject may receive about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 , 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 2 A therapeutic amount of iRNA, such as 1, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg, may be administered. Values and ranges intermediate to the recited values are also contemplated as part of the invention.

In certain embodiments of the invention, for example, when a double-stranded RNAi agent includes a modification (e.g., one or more motifs of three identical modifications on three consecutive nucleotides), e.g., one such motif at or near the cleavage site of the agent, six phosphorothioate linkages, and a ligand, such an agent may be administered at a dose of about 0.01 to about 0.5 mg/kg, about 0.01 to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09 mg/kg, about 0.01 mg/kg to about 0.08 mg/kg, about 0.01 mg/kg to about 0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4 mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg, about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg, about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07 mg/kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about 0.05 mg/kg, about 0 0.03 to about 0.5 mg/kg, about 0.03 to about 0.4 mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0.09 mg/kg, about 0.03 mg/kg to about 0.08 mg/kg, about 0.03 mg/kg to about 0.07 mg/kg, about 0.03 mg/kg to about 0.06 mg/kg, about 0.03 mg/kg to about 0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4 mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 to about 0.2 mg/kg, about 0.04 about 0.05 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg, about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07 mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5 mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg, about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05 mg/kg to about 0.09 mg/kg, about 0.05 mg/kg to about 0.08 mg/kg, or about 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate to the above recited values are also contemplated as part of the invention, for example, the RNAi agent may be administered to a subject at a dose of about 0.015 mg/kg to about 0.45 mg/kg.

For example, the RNAi agent, e.g., the RNAi agent in a pharmaceutical composition, may be about 0.01 mg/kg, 0.0125 mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg, 0.025 mg/kg, 0.0275 mg/kg, 0.03 mg/kg, 0.0325 mg/kg, 0.035 mg/kg, 0.03 75mg/kg, 0.04mg/kg, 0.0425mg/kg, 0.045mg/kg, 0.0475mg/kg, 0.05mg/kg, 0.0525mg/kg, 0.055mg/kg, 0.0575mg/kg, 0.06mg/kg, 0.0625mg/kg, 0 .065mg/kg, 0.0675mg/kg, 0.07mg/kg, 0. 0725mg/kg, 0.075mg/kg, 0.0775mg/kg, 0.08mg/kg, 0.0825mg/kg, 0.085mg/kg, 0.0875mg/kg, 0.09mg/kg, 0.0925mg/kg, 0.095mg/kg, 0.0975mg/k g, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg, 0. It may be administered at a dose of 175 mg/kg, 0.2 mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg, 0.325 mg/kg, 0.35 mg/kg, 0.375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45 mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to the aforementioned recited values are also intended to be part of the invention.

The iRNA may be administered by intravenous infusion, such as over 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 minutes. Administration may be repeated periodically, such as weekly or biweekly (i.e., every two weeks), for one, two, three, four, or more months. After an initial treatment regimen, treatment may be administered less frequently, such as weekly or biweekly for three months, followed by monthly administration for six months or more.

Administration of an iRNA may, for example, increase APOC3 levels in a patient's cells, tissues, blood, urine, or other compartment by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 105%, 106%, 107%, 108%, 109%, 109%, 110%, %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

Prior to administration of the full dose of iRNA, the patient may be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as allergic reactions. In another example, the patient may be monitored for undesired immunostimulatory effects, such as increased levels of cytokines (e.g., TNF-α or IFN-α).

Due to their inhibitory effect on APOC3 expression, the composition according to the present invention or a pharmaceutical composition prepared therefrom may improve quality of life.

The iRNA of the present invention may be administered in a "naked" form, where the modified or unmodified iRNA agent is directly suspended as a "free iRNA" in an aqueous solvent or a suitable buffer solvent. The free iRNA is administered in the absence of a pharmaceutical composition. The free iRNA may be in a suitable buffer. The buffer may include acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS). The pH and osmolality of the buffer containing the iRNA can be adjusted to be suitable for administration to a subject.

Alternatively, the iRNA of the present invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects who may benefit from reducing and/or inhibiting APOC3 gene expression are those who have an APOC3-related disease or disorder as described herein.

Treatment of subjects who may benefit from reducing and/or inhibiting APOC3 gene expression includes therapeutic and prophylactic treatments.

The invention further provides methods and uses of iRNA agents or pharmaceutical compositions thereof in combination with other pharmaceutical agents and/or other therapies, e.g., known pharmaceutical agents and/or known therapies, e.g., those currently used to treat these diseases, to treat subjects who may benefit from reduced and/or inhibited APOC3 expression, e.g., subjects having an APOC3-associated disease. For example, in certain embodiments, an iRNA targeting APOC3 is administered in combination with an additional agent useful, e.g., in the treatment of an APOC3-associated disease.

Examples of additional therapeutic agents include those known to treat hypertriglyceridemia and other disorders caused by, associated with, or resulting from hypertriglyceridemia. For example, iRNAs featured in the invention can be used with, or in combination with, HMG-CoA reductase inhibitors (e.g., statins), fibrates, bile acid sequestrants, niacin, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists (e.g., Merck & Co., Inc., 2002). Co.), acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTTP) inhibitors, cholesterol modulators, bile acid modulators, peroxisome proliferator-activated receptor (PPAR) agonists, gene-based therapies, combination vascular protectants (e.g., AGI-1067 from Atherogenics), glycoprotein Ilb/IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors (e.g., S-8921 from Shionogi), squalene synthase inhibitors, monocyte chemotactic protein (MCP)-I inhibitors, or fish oil. Exemplary HMG-CoA reductase inhibitors include atorvastatin (Lipitor®/Tahor/Sortis/Torvast/Cardyl from Pfizer), pravastatin (Pravachol from Bristol-Myers Squibb, Mevalotin/Sanaprav from Sankyo), simvastatin (Zocor®/Sinvacor from Merck, Denan from Boehringer Ingelheim, Lipovas from Banyu), lovastatin (Mevacor/Mevinacor from Merck, Lovastatina, Cepa from Bexal; Schwarz Pharma's Liposcler), fluvastatin (Novartis's Lescol®/Locol/Lochol, Fujisawa's Cranoc, Solvay's Digaril), cerivastatin (Bayer's Lipobay/GlaxoSmithKline's Baycol), rosuvastatin (AstraZeneca's Crestor®), and pitivastatin (itavastatin/risivastatin) (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis). Exemplary fibrates include, for example, bezafibrate (e.g., Befizal®/Cedur®/Bezali® from Roche, Bezatol from Kissei), clofibrate (e.g., Atromid-S® from Wyeth), fenofibrate (e.g., Lipidil/Lipantil from Fournier, Tricor® from Abbott, Lipantil from Takeda, generics), gemfibrozil (e.g., Lopid/Lipur from Pfizer), and ciprofibrate (Modalim® from Sanofi-Synthelabo). Exemplary bile acid sequestrants include, for example, cholestyramine (Questran® and Questran Light™ from Bristol-Myers Squibb), colestipol (e.g., Colestid from Pharmacia), and colesevelam (WelChol™ from Genzyme/Sankyo). Exemplary niacin therapies include, for example, immediate release preparations such as Nicobid from Aventis, Niacor from Upsher-Smith, Nicolar from Aventis, and Perycit from Sanwakagaku. Niacin extended release formulations include, for example, Niaspan from Kos Pharmaceuticals and SIo-Niacin from Upsher-Smith. Exemplary antiplatelet agents include, for example, aspirin (e.g., Aspirin from Bayer), clopidogrel (Plavix from Sanofi-Synthelabo/Bristol-Myers Squibb), and ticlopidine (e.g., Ticlid from Sanofi-Synthelabo and Panaldine from Daiichi). Other aspirin-like compounds useful in combination with dsRNA targeting APOC3 include, for example, Asacard (Pharmacia's extended release aspirin) and Pamicogrel (Kanebo/Angelini Ricerche/CEPA).Exemplary angiotensin-converting enzyme inhibitors include, for example, ramipril (e.g., Aventis's Altace) and enalapril (e.g., Merck & Co.'s Vasotec). Exemplary acyl-CoA cholesterol acetyltransferase (AC AT) inhibitors include, for example, avasimibe (Pfizer), eflucimibe (BioMsrieux Pierre Fabre/Eli Lilly), CS-505 (Sankyo and Kyoto), and SMP-797 (Sumito). Exemplary cholesterol absorption inhibitors include, for example, ezetimibe (Merck/Schering-Plough Pharmaceuticals Zetia®) and Pamaqueside (Pfizer). Exemplary CETP inhibitors include, for example, torcetrapib (also referred to as CP-529414, Pfizer), JTT-705 (Japan Tobacco), and CETi-I (Avant Immunotherapeutics). Exemplary microsomal triglyceride transfer protein (MTTP) inhibitors include, for example, implitapide (Bayer), R-103757 (Janssen), and CP-346086 (Pfizer). Other exemplary cholesterol modulators include, for example, NO-1886 (Otsuka/TAP Pharmaceutical), CI-1027 (Pfizer), and WAY-135433 (Wyeth-Ayerst).

Exemplary bile acid modulators include, for example, HBS-107 (Hisamitsu/Banyu), Btg-511 (British Technology Group), BARI-1453 (Aventis), S-8921 (Shionogi), SD-5613 (Pfizer), and AZD-7806 (AstraZeneca). Exemplary peroxisome proliferator-activated receptor (PPAR) agonists include, for example, tesaglitazar (AZ-242) (AstraZeneca), netoglitazone (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand Pharmaceuticals and Eli Lilly), and LY-465608 (Ligand Pharmaceuticals and Eli Lilly). Lilly), LY-518674 (Ligand Pharmaceuticals and Eli Lilly), and MK-767 (Merck and Kyorin). Exemplary gene-based therapeutics include, for example, AdGWEGF 121.10 (GenVec), ApoA1 (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark Therapeutics), and ATP-binding cassette transporter-A1 (ABCA1) (CV Therapeutics/Incyte, Aventis, Xenon). Exemplary glycoprotein Ilb/IIIa inhibitors include, for example, roxifiban (also referred to as DMP754, Bristol-Myers Squibb), gantofiban (Merck KGaA/Yamanouchi), and chromafiban (Millennium Pharmaceuticals). Exemplary squalene synthase inhibitors include, for example, BMS-1884941 (Bristol-Myers Squibb), CP-210172 (Pfizer), CP-295697 (Pfizer), CP-294838 (Pfizer), and TAK-475 (Takeda). Exemplary MCP-I inhibitors include, for example, RS-504393 (Roche Bioscience). The anti-atherosclerotic agent BO-653 (Chugai Pharmaceuticals), and the nicotinic acid derivative Nyclin (Yamanouchi Pharmaceuticals) are also suitable for administration in combination with the dsRNA featured in the invention. Exemplary combination therapeutic agents suitable for administration with dsRNA targeting APOC3 include, for example, advicor (niacin/lovastatin from Kos Pharmaceuticals), amlodipine/atorvastatin (Pfizer), and ezetimibe/simvastatin (e.g., Vytorin® 10/10, 10/20, 10/40, and 10/80 tablets from Merck/Schering-Plough Pharmaceuticals). Suitable agents for administration in combination with dsRNA targeting APOC3 for the treatment of hypertriglyceridemia include, for example, lovastatin, niacin Altoprev® extended release tablets (Andrx Pharmaceuticals). Labs), lovastatin Caduet® tablets (Pfizer), amlodipine besylate, atorvastatin calcium Crestor® tablets (AstraZeneca), rosuvastatin calcium Lescol® capsules (Novartis), fluvastatin sodium Lescol® (Reliant, Novartis), fluvastatin sodium Lipitor® tablets (Parke-Davis), atorvastatin calcium Lofibra® capsules (Gate), Niaspan extended release tablets (Kos), niacin Pravachol tablets (Bristol-Myers Squibb), pravastatin sodium TriCor® tablets (Abbott), fenofibrate Vytorin® 10/10 tablets (Merck/Schering-Plough Pharmaceuticals), ezetimibe, simvastatin WelChol™ tablets (Sankyo), colesevelam hydrochloride Zetia® tablets (Schering), ezetimibe Zetia® tablets (Merck/Schering-Plough Pharmaceuticals), and ezetimibe Zocor® tablets (Merck).

In one embodiment, the iRNA agent is administered in combination with an ezetimibe/simvastatin combination (e.g., Vytorin® (Merck/Schering-Plough Pharmaceuticals)). In one embodiment, the iRNA agent is administered to a patient and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered simultaneously.

The iRNA agent and the additional therapeutic agent and/or treatment may be administered simultaneously and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times, and/or by other methods known in the art or described herein.

The invention also provides methods of reducing and/or inhibiting APOC3 expression in a cell using an iRNA agent of the invention and/or a composition containing an iRNA agent of the invention. In another aspect, the invention provides an iRNA of the invention and/or a composition containing an iRNA of the invention for use in reducing and/or inhibiting APOC3 expression in a cell. In yet another aspect, the invention provides the use of an iRNA of the invention and/or a composition containing an iRNA of the invention for the manufacture of a medicament for reducing and/or inhibiting APOC3 expression in a cell.

The methods and uses include contacting a cell with an iRNA, e.g., a dsRNA, of the invention and maintaining the cell for a period of time sufficient to achieve degradation of an mRNA transcript of the APOC3 gene, thereby inhibiting expression of the APOC3 gene in the cell.

Reduction in gene expression can be assessed by any method known in the art. For example, reduction in expression of APOC3 can be determined by determining APOC3 mRNA expression levels using methods routine to one of skill in the art, e.g., Northern blotting, qRT-PCR, determining APOC3 protein levels using methods routine to one of skill in the art, e.g., Western blotting, immunological techniques, flow cytometry, ELISA, and/or determining APOC3 biological activity.

In the methods and uses of the present invention, the cells may be contacted in vitro or in vivo, i.e. the cells may be within the subject's body.

A cell suitable for treatment with the methods of the invention can be any cell that expresses the APOC3 gene. A cell suitable for use in the methods and uses of the invention can be a mammalian cell, for example a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), an avian cell (such as a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, for example a human hepatocyte.

APOC3 expression is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109 %, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% inhibition.

The in vivo methods and uses of the invention can include administering to a subject a composition containing an iRNA, where the iRNA comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of the APOC3 gene of the mammal being treated. When the organism being treated is a human, the composition can be administered by any means known in the art, including, but not limited to, subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, such as intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

In some embodiments, administration is via a depot injection. A depot injection may release the iRNA in a consistent manner over an extended period of time. Thus, a depot injection may reduce the frequency of dosing required to achieve a desired effect, e.g., a desired APOC3 inhibition, or therapeutic or prophylactic effect. A depot injection may also provide a more consistent serum concentration. A depot injection may include a subcutaneous injection or an intramuscular injection. In a preferred embodiment, the depot injection is a subcutaneous injection.

In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump implanted subcutaneously. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, intra-arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The method of administration may be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration may be selected to enhance targeting.

In one aspect, the invention also provides a method for inhibiting expression of the APOC3 gene in a mammal, e.g., a human. The invention also provides a composition comprising an iRNA, e.g., a dsRNA, that targets the APOC3 gene in a mammalian cell for use in inhibiting expression of the APOC3 gene in a mammal. In another aspect, the invention provides the use of an iRNA, e.g., a dsRNA, that targets the APOC3 gene in a mammalian cell in the manufacture of a medicament for inhibiting expression of the APOC3 gene in a mammal.

The methods and uses include administering to a mammal, e.g., a human, a composition comprising an iRNA, e.g., a dsRNA, that targets the APOC3 gene in cells of the mammal, and maintaining the mammal for a time sufficient to achieve degradation of mRNA transcripts of the APOC3 gene, thereby inhibiting expression of the APOC3 gene in the mammal.

Reduction in gene expression can be assessed in peripheral blood samples from subjects administered iRNA by any method known in the art, such as qRT-PCR as described herein. Reduction in protein production can be assessed by any method known in the art and as described herein, such as ELISA or Western blotting. In one embodiment, a puncture liver biopsy sample serves as tissue material for monitoring reduction in APOC3 gene and/or protein expression. In another embodiment, a blood sample serves as tissue material for monitoring reduction in APOC3 gene and/or protein expression.

In one embodiment, confirmation of RISC-mediated in vivo target cleavage following administration of the iRNA agent is performed by performing 5'-RACE or a modified version of a protocol as known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38(3)p-e19) (Zimmermann et al. (2006) Nature 441:111-4).

The present invention is further illustrated by the following examples, which should not be construed as limiting. The entire contents of all references, patents and published patent applications, as well as figures and sequence listings, cited throughout this application are hereby incorporated by reference.

Materials and Methods The following materials and methods were used in this example.

Sources of Reagents If the source of a reagent is not specifically indicated herein, such reagents may be obtained from any molecular biology reagent supplier of quality/purity standards for applications in molecular biology.

siRNA synthesis. APOC3 siRNA sequences were synthesized on a 1 μmol scale using solid support-mediated phosphoramidite chemistry on a Mermade 192 synthesizer (BioAutomation). Solid supports were either controlled pore glass (500 Å) or universal solid supports (AM biochemical) loaded with custom GalNAc ligands. Ancillary synthesis reagents, 2′-F and 2′-O-methyl RNA and deoxyphosphoramidites were obtained from Thermo-Fisher (Milwaukee, WI) and Hongene (China). 2′F, 2′-O-methyl, GNA (glycol nucleic acid), 5′ phosphate and abasic modifications were introduced using the corresponding phosphoramidites. Synthesis of 3′ GalNAc-conjugated single strands was carried out on GalNAc-modified CPG supports. A custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling times for all phosphoramidites (100 mM in acetonitrile) were 5 min using 5-ethylthio-1H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate bonds were created using a 50 mM solution of 3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes, Wilmington, MA, USA) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation times were 3 min. All sequences were synthesized with final removal of the DMT group ("DMT off").

First, the "TOFFEE-6PS" motif was used to synthesize APOC3 sequences for in vitro screening assays. In the TOFFEE-6PS design, a 21 nucleotide long sense strand is created with a GalNAc ligand at the 3' end, two phosphorothioates at the 5' end, and a triplet of 2'F nucleotides at positions 9, 10, and 11. The antisense sequence in the TOFFEE-6PS design is 23 nucleotides long; it contains a three nucleotide triplet of 2'-OMe nucleotides at positions 11, 12, and 13, with two phosphorothioates at each of the 3' and 5' ends.

After completion of the solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and deprotected using 200 μL aqueous methylamine reagent at 60 °C for 20 min in a sealed 96-deep-well plate. For sequences containing 2' ribo residues (2'-OH) protected with tert-butyldimethylsilyl (TBDMS) groups, a second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride) reagent. To this methylamine deprotection solution, 200 μL dimethylsulfoxide (DMSO) and 300 μl TEA.3HF reagent were added and the solution was incubated at 60 °C for another 20 min. Upon completion of the cleavage and deprotection steps, the synthesis plate was brought to room temperature and precipitated by adding 1 mL of acetonitrile:ethanol mixture (9:1). The plate was cooled at -80°C for 2 hours and the supernatant was carefully decanted using a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc buffer and desalted using a 5 mL HiTrap size-exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples were collected in a 96-well plate. Samples from each sequence were analyzed by LC-MS to confirm identity, quantified by UV (260 nm), and determined purity of select sample sets by IEX chromatography.

Annealing of APOC3 single strands was performed on a Tecan liquid handling robot. Equimolar mixtures of sense and antisense single strands were combined and annealed in a 96-well plate. After combining the complementary single strands, the 96-well plate was tightly sealed and heated in an oven at 100°C for 10 minutes and allowed to slowly come to room temperature over a period of 2-3 hours. The concentration of each duplex was standardized to 10 μM in 1x PBS and then subjected to the in vitro screening assay.

Cell Culture and 96-Well Transfection Hep3B cells (ATCC, Manassas, VA) were grown to near confluence in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) at 37°C in an atmosphere of 5% CO2 and then detached from the plate by trypsinization. Transfections were performed by adding 14.8 μl Opti-MEM + 0.2 μl Lipofectamine RNAiMax (Invitrogen, Carlsbad CA. Catalog No. 13778-150) per well to 5 μl of siRNA duplex per well in a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of antibiotic-free complete growth medium containing approximately 2×104 Hep3B cells was then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentrations, and reaction experiments were performed over a dose range of 10 nM to 36 fM final duplex concentrations over eight 6-fold dilutions.

Total RNA isolation using DYNABEADS mRNA isolation kit (Invitrogen; part no. 610-12) Cells were harvested and lysed in 150 μl lysis/binding buffer, then mixed using an Eppendorf Thermomixer at 850 rpm for 5 minutes (mixing speed was the same throughout the run). 10 μl of magnetic beads and 80 μl of lysis/binding buffer mix were placed in a round bottom plate and mixed for 1 minute. The magnetic beads were captured using a magnetic stand and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were placed in the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The beads were captured again and the supernatant removed. The beads were then washed with 150 μl of wash buffer B, captured and the supernatant removed. The beads were then washed with 150 μl of elution buffer, captured and the supernatant removed. The beads were allowed to dry for 2 minutes. After drying, 50 μl of elution buffer was added and mixed for 70 minutes at 5° C. The beads were captured on a magnet for 5 minutes. 40 μl of the supernatant was removed and placed into another 96-well plate.

cDNA synthesis using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Catalog No. 4368813) A master mix of 2 μl 10× buffer, 0.8 μl 25× dNTPs, 2 μl random primers, 1 μl reverse transcriptase, 1 μl RNase inhibitor, and 3.2 μl HO was added to 10 μl total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) with the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real-time PCR
2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan probe (Applied Biosystems Cat. No. 4326317E), 0.5 μl ApoC3 TaqMan probe (Applied Biosystems Cat. No. Hs00163644_m1) and 5 μl Lightcycler 480 Probe Master Mix (Roche Cat. No. 04887301001) per well in a 384-well plate (Roche Cat. No. 04887301001). Real-time PCR was performed using the ΔΔCt (RQ) assay on a LightCycler 480 Real-time PCR system (Roche). Unless otherwise noted in the summary table, each duplex was tested in two independent transfections and each transfection was assayed in duplicate.

To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with 10 nM AD-1955-transfected or mock-transfected cells. IC50s were calculated using a four-parameter fit model using XLFit and normalized to AD-1955-transfected or naive cells.

The sense and antisense sequences of AD-1955 are as follows:
Sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 28)
Antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 29)

Example 1. iRNA Synthesis Generation of an Initial Set of iRNA Agents Targeting APOC3 A set of iRNAs targeting human APOC3, "apolipoprotein C-III" (human: NCBI reference sequence number NM_000040.1), as well as tox species APOC3 orthologues (cynomolgus monkey: XM_005579730; rhesus monkey, XM_001090312.2; mouse: NM_023114; rat, NM_012501) were designed using custom R and Python scripts. The human APOC3 REFSEQ mRNA has a length of 533 bases. The rationale and methodology for this set of siRNA designs are as follows: The predicted efficacy of every possible 19mer iRNA from positions 47-533 was determined with a linear model derived from direct measurement of mRNA knockdown from over 20,000 different iRNA designs targeting multiple vertebrate genes. A subset of APOC3 siRNAs was designed with perfect or near perfect matches between human, cynomolgus and rhesus monkeys. An additional subset was designed with perfect or near perfect matches to mouse and rat APOC3 orthologues. For each strand of the iRNA, a custom Python script was used in a brute force search to determine the number and location of mismatches between the siRNA and every possible alignment in the target species transcriptome. Additional weight was added to mismatches in the seed region (defined here as positions 2-9 of the antisense oligonucleotide) as well as the cleavage site of the iRNA (defined here as positions 10-11 of the antisense oligonucleotide). The relative weights of the mismatches were 2.8; 1.2:1 for the seed mismatch, cleavage site, and other positions up to antisense position 19. Mismatches at the first position were ignored. A specificity score was calculated by summing the values of each weighted mismatch for each strand. Priority was given to selecting siRNAs with an antisense score >=3.0 in humans and cynomolgus monkeys and predicted efficacy >=70% knockdown of APOC3 transcripts.

A series of iRNA duplexes with sequences designed as described above were synthesized and conjugated with trivalent GalNAc at the 3' end of the sense strand using the techniques described above. The sequences of these duplexes are shown in Tables 4A and 4B. These same sequences with various nucleotide modifications were also synthesized and conjugated with trivalent GalNAc. The sequences of the modified duplexes are shown in Table 5.

Example 2. In Vitro Testing of siRNA Sequences Table 6 shows the results of a single dose screen in Hep3B cells using selected modified APOC3 iRNAs. Data are expressed as the percentage of APOC3 mRNA remaining in cells transfected with the iRNA relative to the percentage of APOC3 mRNA remaining in cells transfected with the AD-1955 non-targeting control.

Table 7 shows the dose response of Hep3B cells transfected with the indicated cynomolgus/human cross-reactive modified APOC3 iRNAs. The indicated IC50 values represent the IC50 values relative to untreated cells. The results of the APOC3 iRNA single dose screen (10 nM mean value blue bar and 0.1 nM mean value red bar) are shown in Figure 1. Based on the results of this screen, three iRNAs (AD-57553.1, AD-57547.1, and AD-58924.1) were selected for further in vivo testing.

Example 3. In vivo testing of AD-57558 siRNA sequences in wild-type mice. The rodent-specific AD-57558-GalNAc3 sequence was tested for its ability to inhibit APOC3 expression and reduce serum lipids in wild-type mice. AD-57558-GalNAc3 was administered subcutaneously at a single dose of 3 mg/kg, 10 mg/kg, or 30 mg/kg, and PBS was used as a negative control. APOC3 expression was measured by RT-PCR 5 days after administration. The results shown in FIG. 2 indicate that AD-57558-GalNAc3 can reduce APOC3 expression by about 60% (for the 3 mg/kg dose), about 80% (for the 10 mg/kg dose), and about 85% (for the 30 mg/kg dose).

Example 4. Creation and characterization of APOC3 overexpression mouse model To enable in vivo ApoC3 GalNAc conjugation lead discovery with compounds that do not cross-react with the rodent APOC3 gene, an overexpression system of human APOC3 gene in the liver of C57Bl/6 mice was used. An adeno-associated virus serotype 8 (AAV8) encapsidated AAV2 vector genome expressing human APOC3 under the liver-specific TBG promoter was created. Model characterization studies were performed to identify optimal conditions, including viral genome copy (GC) number and time required for high and sustained expression of human ApoC3 in AAV-transduced mice.

Further studies were performed using administration of 10 11 genome copies of the AAV2 vector per mouse. APOC3 mRNA levels were measured by RT-PCR in the livers of AAV-hApoC3 mice 1.5, 6.5, 8, and 16 weeks after administration of the hAPOC3 AAV vector. RT-PCR results (data not shown) indicate that high expression levels of the human APOC3 gene were achieved in mice within 10 days after administration of 10 11 hAPOC3 AAV genome copies and persisted for at least 6 months, with little inter-animal variability.

Example 5. Testing Potential Lead iRNAs in an APOC3 Overexpressing Mouse Model Following characterization, the APOC3-AAV mouse model system of APOC3 overexpression was used for in vivo screening of AD-57553, AD-57547, and AD-58924, which were identified based on in vitro potency. For the single high dose screen experiments, APOC3-AAV mice pre-injected with 10 hAPOC3 AAV genome copies were administered a single 10 mg/kg dose of AD-57553, AD-57547, and AD-58924 or PBS (as a control) followed by measurement of APOC3 mRNA. Results are provided in Figures 3 and 4. Specifically, Figure 3 shows APOC3 mRNA levels measured in individual APOC3-AAV mice injected with AD-57553, AD-57547, and AD-58924 or PBS, while Figure 4 shows the mRNA group averages. The data indicate that AD-57553 was the most effective at inhibiting APOC3 expression.

AD-57553 was selected for further in vivo testing in dose-response and multiple dose studies. For dose-response experiments, APOC3-AAV mice previously injected with 1011 hAPOC3 AAV genome copies were administered 1.25 mg/kg, 2.5 mg/kg and 5 mg/kg doses weekly for 4 weeks, followed by measurement of APOC3 mRNA levels. Results are provided in Figures 5 and 6. Specifically, Figure 5 shows APOC3 mRNA levels measured in individual APOC3-AAV mice injected with AD-57553, while Figure 6 shows the mRNA group average. These data demonstrate increasing inhibition of APOC3 mRNA expression with increasing doses of AD-57553.

Example 6. Generation and in vivo testing of additional RNA sequences based on AD-57553 From the initial round of SAR optimization of iRNA chemistry, ten additional iRNA sequences were generated based on the AD-57553 lead sequence by introducing changes in 2'F, 2'OMe and 5'P modifications. The additional iRNAs are provided in Tables 8 and 9 below.

These additional sequences were tested for their ability to inhibit APOC3 protein expression in APOC3-AAV mice. Specifically, APOC3-AAV mice previously injected with 1011 hAPOC3 AAV genome copies were administered a single dose of 3 mg/kg of the indicated modified iRNA and the resulting circulating serum APOC3 protein levels were measured 5, 10 and 20 days after administration. Figure 7A provides the time course of serum APOC3 protein levels measured for each iRNA sequence tested up to day 20, and Figure 7B provides the time course of serum APOC3 protein levels for six selected iRNA sequences up to day 30. Figures 8 and 9 represent the data for days 10 and 20, respectively, for each iRNA sequence tested. These data indicate that the most active iRNA sequences, such as AD-65704, are able to achieve approximately 80% knockdown of serum APOC3 protein at days 10 and 20.

Example 7. Testing of Novel Lead iRNAs in an APOC3 Overexpressing Mouse Model AD-57553, AD-65696, AD-65703 and AD-65704 were selected for follow-on studies to test the effect of fluorine content and vinyl phosphate on the ability of the iRNA agents to inhibit expression of APOC3 protein in vivo. Table 9 above shows the modified sequences of AD-57553, AD-65696, AD-65703 and AD-65704, and Table 10 below includes a brief description of the modifications present in each strand.

The ability of iRNAs to inhibit human APOC3 protein expression in vivo was tested in the APOC3-AAV mouse model system of APOC3 overexpression. For the single dose screen study, APOC3-AAV mice pre-injected with 10 APOC3 AAV genome copies were administered a single 3 mg/kg dose of AD-57553, AD-65696, AD-65703, and AD-65704 or PBS (as a control) and APOC3 protein levels were measured at 0, 3, 10, 20, 30, 41, 55, and 70 days post-treatment compared to pre-treatment. Figure 10 provides data for all four iRNAs tested. The data in Figure 10 show that iRNAs with low 2'F content (AD-65703 and AD-65704) are able to maintain approximately 80% knockdown of APOC3 over a 30-day period. The data also demonstrate that after a single 3 mg/kg dose, APOC3 levels take 55-70 days to return to those observed in the control (PBS) group.

AD-57553, AD-65696, AD-65699, AD-65703 and AD-65704 were used for further in vivo testing in dose-response and multiple dose studies. For dose-response experiments, APOC3-AAV mice pre-injected with 1011 APOC3 AAV genome copies received four subcutaneous doses of 0.3 mg/kg, 1 mg/kg and 3 mg/kg of each iRNA (Q2Wx4 dosing schedule). Animals were bled on days 0, 7, 14, 21, 35, 49, 62 and 77 to assess APOC3 loading. The dosing schedule used for this experiment is illustrated diagrammatically in Figure 11.

The time courses for the 0.3 mg/kg, 1 mg/kg and 3 mg/kg doses are shown in Figures 12A, 12B and 12C, respectively. The data in Figures 12A-12C demonstrate that at the 0.3 mg/kg dose, each of the tested iRNAs is able to inhibit APOC3 protein expression to 50% of pre-dosing measurements. These data also demonstrate that at the 1 mg/kg and 3 mg/kg doses, AD-65699, which does not contain a 2'F modification, is less effective at inhibiting APOC3 expression than the other iRNAs tested. These data further demonstrate that up to a 94% reduction in relative APOC3 levels is achieved after animals are dosed with a 3 mg/kg dose of AD-57553, AD-65696, AD-65703 and AD-65704. This knockdown of APOC3 levels persists for at least 3 weeks after the final dose. Two of the iRNAs tested, AD-65696 and AD-65704, were able to maintain approximately 80% inhibition of APOC3 protein for at least five weeks after the final dose.

AD-65704, which contains six 2'F modifications on one of the selected lead sequences, the sense strand and the antisense strand, was tested in a single dose titration study using the APOC3-AAV mouse model system of APOC3 overexpression. For the dose screen experiment, APOC3-AAV mice pre-injected with 1011 hAPOC3 AAV genome copies were administered a single dose of 0.3 mg/kg, 1 mg/kg or 3 mg/kg AD-65704 or PBS (as a control) and APOC3 protein levels were measured 14 days later compared to pre-administration. The results provided in Figure 13 show that the effective dose to achieve 80% inhibition of APOC3 expression (ED80) is approximately 3 mg/kg, while the effective dose to achieve 40% inhibition of APOC3 expression (ED40) is 1 mg/kg.

For dose titration experiments, AD-65704 was administered subcutaneously at doses of 0.3 mg/kg, 1 mg/kg, or 3 mg/kg to APOC3-AAV mice previously injected with 1011 APOC3 AAV genome copies. Each dose level was administered once every other week for a total of four doses (Q2W x 4 doses). Figure 14 shows the amount of APOC3 protein measured 20 days after the last dose compared to pre-dose. These data indicate that the effective dose to achieve 90% inhibition of APOC3 expression (ED90) is ≦3 mg/kg; the effective dose to achieve 70% inhibition of APOC3 expression (ED70) is 1 mg/kg; and the effective dose to achieve 50% inhibition of APOC3 expression (ED50) is 0.3 mg/kg.

The results provided in this example (Figures 10-14) demonstrate that low fluorine content iRNAs (AD-65703 and AD-65704) achieve approximately 80% knockdown of APOC3 when administered as a single 3 mg/kg dose that is sustained for at least 30 days (see Figure 10). Four doses of 3 mg/kg administered every 2 weeks achieve up to 94% reduction in APOC3 levels with >90% knockdown sustained for at least 3 weeks (see Figure 12C).

Results with ultra-low fluorine content iRNA (AD-65698) show that this iRNA can achieve up to 83% knockdown with four doses of 3 mg/kg administered every 2 weeks, with 75% knockdown sustained for 3 weeks after the final dose (see Figure 12C). Results with fluorine-free iRNA (AD-65699) show that this iRNA can achieve extremely short-term knockdown with a single dose of 3 mg/kg. Four doses of 3 mg/kg administered every 2 weeks can increase knockdown by up to 50%, with APOC3 levels returning to baseline within 2 weeks after administration of the final dose (see Figure 12C).

Addition of VP to the 5' of the antisense strand boosts the ability of the iRNA to inhibit APOC3 expression. This is evident from the 94% APOC3 knockdown observed after a single 3 mg/kg dose of VP-containing AD-65696 compared to the approximately 80% knockdown observed with the parent iRNA AD-57553 without VP (see Figure 10). Multiple dose experiments with AD-65696 resulted in up to 99% knockdown of APOC3 at 3 mg/kg, approximately 80% knockdown at 1 mg/kg, and over 50% knockdown at a 0.3 mg/kg dose (see Figures 12A-C).

Example 8. Identification of iRNAs that cross-react with rabbit APOC3 The iRNA agents shown in Table 7 were analyzed for their ability to cross-react with rabbit APOC3. Based on the number of mismatches with rabbit APOC3, AD-58911, AD-58924, AD-58922 and AD-58916 were determined to have cross-reactivity with rabbit APOC3.

The four rabbit cross-reactive sequences were modified to contain a total of 10 2'F modifications in the sense and antisense strands to yield iRNAs AD-67221, AD-67222, AD-67223, and AD-67224. The unmodified and modified sequences for AD-67221, AD-67222, AD-67223, and AD-67224 are provided below in Tables 11A and 11B, respectively.

These iRNAs were tested in a single-dose study using the APOC3-AAV mouse model system of APOC3 overexpression. APOC3-AAV mice pre-injected with 10 hAPOC3 AAV genome copies were administered a single dose of 1 mg/kg AD-65704, AD-67221, AD-67222, AD-67223, AD-67224, or PBS (as a control) and APOC3 protein levels were measured 14 and 26 days after treatment compared to pre-treatment. The results presented in FIG. 15 show that at day 14, iRNAs AD-67222 and AD-67224 were inactive at 1 mg/kg, AD-67223 showed 30% inhibition of APOC3 protein, and AD-67221 was comparable to AD-65704 with approximately 40% inhibition of APOC3 protein at the 1 mg/kg dose. Initial silencing of APOC3 protein is similar between AD-65704 and AD-67221 at day 14, but activity levels at day 26 show that AD-65704 is more sustained than AD-67221 (achieving 46% and 33% inhibition of APOC3 protein, respectively).

Example 9. Testing the effect of vinyl phosphate modifications The purpose of this study was to test the effect of vinyl phosphate (VP) and 2'F modifications on the antisense strand on the ability of iRNAs to inhibit APOC3 expression. The iRNAs used in this study are summarized in Table 12 below.

iRNA agents AD-65698 and AD-66239 have the same sense strand but different antisense strands. Similarly, duplexes AD-65701 and AD-66240 have the same sense strand but different antisense strands. iRNA agents AD-65704 and AD-66241 contain the same sense strand but different antisense strands. The antisense strands of AD-65698, AD-65701 and AD-65704 are the same and contain four nucleotides that contain phosphorothioates and two nucleotides that contain 2'-fluoro modifications. The antisense strands of AD-66239, AD-66240 and AD-66241 are the same and contain four nucleotides that contain phosphorothioates, nine nucleotides that contain 2'-fluoro modifications, and one vinyl phosphate at the 5' end of the antisense strand.

APOC3-AAV mice pre-injected with 1011 APOC3 AAV genome copies were administered a single 1 mg/kg dose of each iRNA listed in Table 11 or PBS (as a control), and APOC3 protein levels were measured 10 and 24 days after administration compared to pre-administration. Data from day 10 are shown in Figure 16A, and data from day 24 are shown in Figure 16B.

These results demonstrate that mixed matching of VP modifications on the antisense strand with low fluorine content in the sense strand resulted in a similar boost in activity, however, failed to improve duration compared to pairing the sense strand with an antisense strand without a VP modification.

Example 10. In vivo testing of additional iRNA sequences based on AD-65704 SAR optimization of AD-65704 chemistry yielded 10 additional iRNA sequences (Table 13).

These iRNAs were tested in a single-dose study using the APOC3-AAV mouse model system of APOC3 overexpression. Briefly, 2 weeks after APOC3-AAV mice were injected with 10 hAPOC3 AAV genome copies, mice (n=3) were subcutaneously administered a single 1 mg/kg dose of iRNA agent or PBS (as a control). At 14, 28, and 42 days post-dose, serum APOC3 protein levels were measured by ELISA assay relative to pre-dose levels. The results, presented in Figures 17A and 17B, show that at day 14, all of the tested iRNAs were active at 1 mg/kg.

Example 11. In vivo testing of iRNA agents in non-human primates Based on the results described in Example 10, three iRNA agents, AD-65704, AD-69535 and AD-69541, were selected for evaluation in non-human primates (primtaes).

Single and multiple dose experiments were performed in cynomolgus monkeys. In one set of experiments, naive male cynomolgus monkeys (n=3) were given a single weekly dose of 1 mg/kg AD-65704 subcutaneously on day 1, or naive male cynomolgus monkeys (n=3) were given weekly doses of 1 mg/kg AD-65704 subcutaneously on days 1, 8, 15, 22, 29, 36, 43, and 50. Serum was collected on days -7, -1, 1, 8, 11, 15, 22, 29, 36, 43, 57, 64, and 71. Cynomolgus ApoC3 protein levels were determined by ELISA. Liver biopsies were performed on days -7, 12, 30, and 64 to determine ApoC3 mRNA levels. The results of the single dose study are illustrated in Figures 18A and 18B and demonstrate that weekly dosing of 1 mg/kg AD-65704 achieves a >80% reduction in total serum ApoC3 and up to a 50% reduction in total ApoC3 protein (Figure 18B). These data also demonstrate that weekly dosing of 1 mg/kg AD-65704 achieves a 60% reduction in liver ApoC3 mRNA compared to pre-dose levels. As illustrated in Figure 18C, weekly dosing of 1 mg/kg AD-65704 reduces ApoC3 mRNA levels by 95% compared to pre-dose levels.

In another set of experiments, naive male cynomolgus monkeys (n=3) were administered a single weekly 1 mg/kg dose of AD-65704, AD-69535, or AD-69541 subcutaneously on day 1. Serum was collected on days -7, -1, 1, 8, 11, 15, 22, 29, and 36. Cynomolgus ApoC3 protein levels were determined by ELISA. Liver biopsies were performed and ApoC3 mRNA levels were determined on days -7, 12, 30, and 64. These data demonstrate that a single 1 mg/kg dose of all three iRNA agents tested reduced ApoC3 protein levels to approximately 50% of baseline levels (ED50 protein = approximately 1 mg/kg) (Figure 19A). As shown in FIG. 19B, a single 1 mg/kg dose of AD-65704 reduces ApoC3 mRNA by 60% compared to pre-dose levels, AD-69535 reduces ApoC3 mRNA by 68% compared to pre-dose levels, and AD-69541 reduces ApoC3 mRNA by 64% compared to pre-dose levels (ED50 mRNA < 1 mg/kg).

Further multiple dose studies were performed with AD-65704, AD-69535, and AD-69541 in cynomolgus monkeys. Naive male cynomolgus monkeys were administered a single 1 mg/kg dose of AD-65704, AD-69535, or AD-69541 subcutaneously on day 1, followed by a single 3 mg/kg dose of the same agent subcutaneously on day 36. N=3/group. Serum was collected on days -7, -1, 1, 8, 11, 15, 22, 29, 36, 43, 50, 57, 64, and 71. Cynomolgus ApoC3 protein levels were determined by ELISA. Liver biopsies were performed on days -7, 12, 30, and 64 to determine ApoC3 mRNA levels. FIG. 20A demonstrates that administration of a 3 mg/kg dose of AD-65704 or AD-69535 reduces ApoC3 protein levels by approximately 70% compared to pre-dose levels (ED70 protein=approximately 3 mg/kg), and FIG. 20B demonstrates that 64 days after administration of a 3 mg/kg dose of AD-65704, ApoC3 mRNA levels are reduced by 81% compared to pre-dose levels, 64 days after administration of a 3 mg/kg dose of AD-69535, ApoC3 mRNA levels are reduced by 88% compared to pre-dose levels, and 64 days after administration of a 3 mg/kg dose of AD-69541, ApoC3 mRNA levels are reduced by 84% compared to pre-dose levels (ED80 mRNA≦3 mg/kg).

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein which equivalents are intended to be encompassed by the following claims.

Claims (34)

A double-stranded RNAi agent for inhibiting expression of apolipoprotein C3 (APOC3) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region,
the sense strand comprises at least 20 contiguous nucleotides from the nucleotide sequence of 5'-GCUUAAAAGGGACAGUAUUCU-3' (SEQ ID NO:81) , and the antisense strand comprises at least 21 contiguous nucleotides from the nucleotide sequence of 5'-AGAAUACUGUCCCUUUUAAGCAA-3' (SEQ ID NO:184) ;
all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a nucleotide modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 2'-deoxy modification;
the sense and antisense strands independently further comprise at least two phosphorothioate internucleotide linkages; and
At least one chain is conjugated to a ligand;
Double-stranded RNAi agents. 2. The double-stranded RNAi agent of claim 1, wherein the sense strand comprises at least two 2'-fluoro nucleotide modifications, at least seven 2'-O-methyl nucleotide modifications, and at least two phosphorothioate internucleotide linkages. The double-stranded RNAi agent of claim 1 or 2, wherein the antisense strand comprises at least nine 2'-O-methyl nucleotide modifications and at least four phosphorothioate internucleotide linkages. The double-stranded RNAi agent of claim 2 or 3, wherein at least two of the 2'-fluoro nucleotide modifications on the sense strand are 2'-fluoro modified guanosine nucleotides at positions 10 and 11 of the sense strand from the 5' end. 5. The double-stranded RNAi agent of any one of claims 2-4, wherein at least one of the 2'-O-methyl nucleotide modifications on the sense strand is a 2'-O-methyl modified adenosine nucleotide at position 6 of the sense strand from the 5' end. 6. The double-stranded RNAi agent of any one of claims 2-5, wherein at least one of the 2'-O-methyl nucleotide modifications on the sense strand is a 2'-O-methyl modified uridine nucleotide at position 18 of the sense strand from the 5' end. 7. The double-stranded RNAi agent of any one of claims 3 to 6, wherein at least one of the 2'-O-methyl nucleotide modifications on the antisense strand is a 2'-O-methyl modified adenosine nucleotide at position 19 of the antisense strand from the 5' end. 8. The double-stranded RNAi agent of any one of claims 3 to 7, wherein at least one of the 2'-O-methyl nucleotide modifications on the antisense strand is a 2'-O-methyl modified uridine nucleotide at position 17 of the antisense strand from the 5' end. 9. The double-stranded RNAi agent of any one of claims 3 to 8, wherein at least two of the 2'-O-methyl nucleotide modifications on the antisense strand are 2'-O-methyl modified cytosine nucleotides at positions 12 and 13 of the antisense strand from the 5' end. 10. The double-stranded RNAi agent of any one of claims 3-9, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end. The double-stranded RNAi agent of any one of claims 1 to 10, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. The double-stranded RNAi agent of any one of claims 1 to 11 , wherein at least one strand comprises a 3' overhang of at least one nucleotide. The double-stranded RNAi agent of any one of claims 1 to 11 , wherein at least one strand comprises a 3' overhang of at least 2 nucleotides. The double-stranded RNAi agent of any one of claims 1 to 13 , wherein the ligand is one or more GalNAc derivatives. 15. The double-stranded RNAi agent of claim 14 , wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. The ligand is

The double-stranded RNAi agent of claim 15 , The double-stranded RNAi agent of any one of claims 1 to 16 , wherein the ligand is attached to the 3' end of the sense strand. Schematic diagram below

The double-stranded RNAi agent of any one of claims 1 to 17 , conjugated to a ligand as shown in the formula: 20. The double stranded RNAi agent of claim 18, wherein X is O. An isolated cell containing the double-stranded RNAi agent of any one of claims 1 to 19 . A pharmaceutical composition comprising the double-stranded RNAi agent of any one of claims 1 to 19 . 22. The pharmaceutical composition of claim 21 , wherein the double-stranded RNAi agent is in a non-buffer solution. 23. The pharmaceutical composition of claim 22 , wherein the non- buffered solution is saline or water. 22. The pharmaceutical composition of claim 21 , wherein the double -stranded RNAi agent is in a buffer. 25. The pharmaceutical composition of claim 24 , wherein the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. 25. The pharmaceutical composition of claim 24 , wherein the buffer is phosphate buffered saline (PBS). 1. An in vitro method for inhibiting apolipoprotein C3 (APOC3) expression in a cell, comprising:
(a) contacting said cell with the double-stranded RNAi agent of any one of claims 1-19 or the pharmaceutical composition of any one of claims 21-26 ; and (b) maintaining the cell resulting from step (a) for a time sufficient to achieve degradation of mRNA transcripts of the APOC3 gene, thereby inhibiting expression of the APOC3 gene in said cell. 27. The double-stranded RNAi agent of any one of claims 1-19 or the pharmaceutical composition of any one of claims 21-26 for use in a method of treating a subject having an apolipoprotein C3 (APOC3) associated disease, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent or pharmaceutical composition . 29. The double-stranded RNAi agent or pharmaceutical composition of claim 28 , wherein the APOC3-related disease is hypertriglyceridemia. 29. The double-stranded RNAi agent or pharmaceutical composition of claim 28, wherein the APOC3-related disease is selected from the group consisting of non -alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes mellitus , hypertension, atherosclerosis and pancreatitis. The double-stranded RNAi agent or pharmaceutical composition of any one of claims 28 to 30 , wherein the double-stranded RNAi agent or pharmaceutical composition is for administration at a dose of 0.01 mg/kg to 10 mg/kg or 0.5 mg/kg to 50 mg/kg. The double-stranded RNAi agent or pharmaceutical composition of any one of claims 28 to 31, wherein the double-stranded RNAi agent or pharmaceutical composition is for subcutaneous administration. The double-stranded RNAi agent or pharmaceutical composition of any one of claims 28 to 32, wherein the double-stranded RNAi agent or pharmaceutical composition is for administration in combination with an additional therapeutic agent. 34. The double-stranded RNAi agent or pharmaceutical composition of claim 33, wherein the additional therapeutic agent is selected from the group consisting of HMG-CoA reductase inhibitors, fibrates, bile acid sequestrants, niacin, antiplatelet agents, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, acyl-CoA cholesterol acetyltransferase (ACAT) inhibitors, cholesterol absorption inhibitors, cholesterol ester transfer protein (CETP) inhibitors, microsomal triglyceride transfer protein (MTTP) inhibitors, cholesterol modulators, bile acid modulators, peroxisome proliferator-activated receptor (PPAR) agonists, gene-based therapies, combined vascular protectants, glycoprotein Ilb/IIIa inhibitors, aspirin or aspirin-like compounds, IBAT inhibitors, squalene synthase inhibitors, monocyte chemoattractant protein (MCP)-I inhibitors, or fish oil.